research topics on gas chromatography and mass spectrometry

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GC-MS Principle, Instrument and Analyses and GC-MS/MS

Gas chromatography alone cannot be used for the identification of unknowns, which is where hyphenation to a mass spectrometer comes in..

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What is gas chromatography mass spectrometry?

Gas chromatography mass spectrometry (GC-MS) consists of two very different analytical techniques: gas chromatography (GC) which is hyphenated (hence uses a hyphen not a forward slash) to mass spectrometry (MS) . Usually, the analytical instrument consists of a gas chromatograph that is hyphenated via a heated transfer line to the mass spectrometer, and the two techniques take place in series. However, some specialist and usually miniature or portable instruments contain the whole GC-MS within a single box.

How does a GC-MS instrument work?

Gc-ms analysis and what the retention time tells you.

GC is a separation science technique that is used to separate the chemical components of a sample mixture and then detect them to determine their presence or absence and/or how much is present. GC detectors are limited in the information that they give; this is usually two-dimensional giving the retention time on the analytical column and the detector response. Identification is based on comparison of the retention time of the peaks in a sample to those from standards of known compounds, analyzed using the same method. However, GC alone cannot be used for the identification of unknowns, which is where hyphenation to an MS works very well. MS can be used as a sole detector , or the column effluent can be split between the MS and GC detector(s).

MS is an analytical technique that measures the mass-to-charge ratio (m/z) of charged particles and therefore can be used to determine the molecular weight and elemental composition, as well as elucidating the chemical structures of molecules. Data from a GC-MS is three-dimensional, providing mass spectra that can be used for identity confirmation or to identify unknown compounds plus the chromatogram that can be used for qualitative and quantitative analysis.

The sample mixture is first separated by the GC before the analyte molecules are eluted into the MS for detection. 1 They are transported by the carrier gas (Figure 1 (1)), which continuously flows through the GC and into the MS, where it is evacuated by the vacuum system (6).

1.   The sample is first introduced into the GC manually or by an autosampler (Figure 1 (2)) and enters the carrier gas via the GC inlet (Figure 1 (3)). If the sample is in the liquid form, it is vaporized in the heated GC inlet and the sample vapor is transferred to the analytical column (Figure 1 (4)).

2.   The sample components, the “analytes”, are separated by their differences in partitioning between the mobile phase (carrier gas) and the liquid stationary phase (held within the column), or for more volatile gases their adsorption by a solid stationary phase. In GC-MS analyses, a liquid stationary phase held within a narrow (0.1-0.25 mm internal diameter) and short (10-30 m length) column is most common.

3.   After separation, which for GC-MS analyses doesn’t require total baseline resolution unless the analytes are isomers, the neutral molecules elute through a heated transfer line (Figure 1 (5)) into the mass spectrometer.

Gas Chromatography – How a Gas Chromatography Machine Works, How To Read a Chromatograph and GCxGC

4.   Within the mass spectrometer, the neutral molecules are first ionized , most commonly by electron ionization (EI) . In EI, an electron, produced by a filament, is accelerated with 70 electron volts (eV) and knocks an electron out of the molecule to produce a molecular ion that is a radical cation. This high energy ionization can result in an unstable molecular ion and excess energy can be lost through fragmentation. Bond breakage(s) can lead to the loss of a radical or neutral molecule and molecular rearrangements can also occur. This all results in a, sometimes very large, number of ions of different masses, the heaviest being the molecular ion with fragment ions of various lower masses, depending on:

• the molecular formula
• the molecular structure of the analyte
• where bond breakage has occurred
• which part has retained the charge

5.   The next step is to separate the ions of different masses, which is achieved based on their m/z by the mass analyzer (Figure 1 (8)).

There are numerous different mass analyzer types, and this is where the vast differences in mass resolution (and hence instrument price) is seen. Mass resolution is the ability of the mass analyzer to separate ions with very small differences in m/z. Unit mass resolution instruments can only separate nominal masses or those down to a single decimal place, whereas high mass resolution (HRMS) instruments can separate them to four or five decimal places.

The most common type of unit mass instrument is the quadrupole , which is a scanning instrument and varies the voltage to allow only ions of a certain m/z to have a stable trajectory through the four poles to reach the ion detector. Quadrupole instruments are used in two different modes of operation:

• Full scan mode , where all ions are acquired across a mass range, useful for identification of unknowns, method development and qualitative and quantitative analysis for higher concentration analytes.
• Selected ion monitoring (SIM) mode, where only selected ions that represent the target compound are acquired, useful for trace analysis, as higher sensitivity is obtained, but only of target analytes.

An ion trap is also a scanning instrument but is three-dimensional, trapping the ions in mass-dependent orbits before ejecting them sequentially to reach the ion detector.

Time-of-flight (ToF) mass analyzers separate the ions based on the time they take to travel down the flight tube to reach the ion detector. With the same kinetic energy, those with lower masses have a higher velocity and therefore arrive first, whereas those with higher masses have a lower velocity and arrive later. ToFMS instruments can range in mass resolution and acquisition rate: very fast ToFs, with acquisition rates of up to 1000 spectra/second are unit mass resolution, whereas HRMS ToFs have a lower acquisition rate. High acquisition rates are good for two-dimensional GC (GC x GC) applications with peak widths down to 30 ms, however HRMS is very useful to determine the molecular formula. Therefore, there are ToFs on the market that range in speed and mass resolution, the choice of which is dependent on the application, but the GC peak width must match the acquisition rate capabilities of the MS.

Other HRMS instruments that are hyphenated to GC include the magnetic sector mass analyzer, which bends the trajectories of the ions to separate them using electric and magnetic fields. Magnetic sector GC-MS instruments are more commonly found in isotope ratio analyses.

In the HRMS orbitrap , the ions orbit around a central spindle and the frequency that they move up and down the central spindle is m/z-dependent.

6.   After the ions have been separated by the mass analyzer based on their m/z, they reach the ion detector (Figure 1 (9)) where the signal is amplified by an electron multiplier (for most low resolution MS) or a multi-channel plate (for most HRMS instruments). The signal is recorded by the acquisition software on a computer (Figure 1 (10)) to produce a chromatogram and a mass spectrum for each data point.

GC-MS data is three-dimensional, as shown in Figure 2. The x-axis shows the retention time; the time from sample injection to the end of the GC run. This can also be viewed as the scan number, which is the number of data points that have been acquired by the MS across the run. The y-axis is the response or intensity measured by the ion detector (Figure 1 (9)) . The z-axis is the m/z of the ions across the mass range acquired.

The two-dimensional chromatogram, as shown in Figure 3, is produced by summing the abundances of all the ions at a single data point and plotting it against the retention time (RT)/scan number to produce a total ion chromatogram (TIC), which is more comparable to a chromatogram produced by a GC detector. However, each data point in the total ion chromatogram is a separate mass spectrum and can usually be opened in a separate window in the software. In the example shown in Figure 3, the apex data point of peak 3 has been opened.

An example mass spectrum of the straight chain hydrocarbon n-decane can be seen in Figure 4. The molecular ion, 142 m/z can be seen on the far right. As decane is a saturated hydrocarbon, the excess energy from ionization cannot be delocalized internally and therefore most molecular ions fragment, resulting in many fragment ions and a low abundance of the molecular ion. The longer the chain length of a saturated hydrocarbon, the lower the abundance of the molecular ion until no molecular ion is observed in the mass spectrum. However, unsaturated molecular ions and especially those with conjugated double bonds, like aromatic compounds, have less fragmentation as the excess energy can be internalized more easily.

Another observation from the mass spectrum of decane is the series of fragment ions at m/z 43, 57, 71, 85, 99 and 113 that differ by an m/z of 14. These are formed by the overlapping cleavage of bonds at successive -C 2 H 4 - units, if the charge is +1 this equates to a mass of 28 unified atomic mass units (u) and is a key feature of mass spectra from hydrocarbons. The mass spectrum is a fingerprint of the molecule and, if obtained using the same ionization technique and voltage, can be compared to libraries of spectra obtained using the same technique at the same voltage. The most common commercial libraries are EI spectra produced at 70 eV. The mass spectrum can also be interpreted to determine the molecular formula and structure of the molecule using the masses of the ions, the presence of isotopes and the losses between the fragment ions.

In GC, the retention time is used for the identification of target analytes and usually the area is used for quantitation. For accurate quantitation, the peaks need to have good chromatographic separation with baseline resolution, as shown in Figure 3 peaks at RT1 and RT2. With GC-MS the mass spectrum provides an additional method for confirmation of the target analyte using the full mass spectrum or using the presence and relative ratios of a few of the ions. Quantitation using GC-MS data is usually from the area of a single, unique ion as it is less likely to have interferences from co-eluting peaks than by using the area under the TIC peak. As such, chromatographic baseline resolution isn’t needed for accurate quantitation, as long as a unique ion can be selected that isn’t present in the coeluting peaks and therefore the peaks are spectrally resolved, and baseline-to-baseline integration can be achieved.

GC-MS vs GC-MS/MS

The use of chromatographic and spectral resolution is very powerful for the separation and identification of target analytes. However, in the analysis of trace analytes down to femtogram (fg) levels in very complex samples, for example environmental, food or biological samples, the matrix can be overwhelming. 2 , 3 Sample preparation can be used to remove the majority of the matrix; however, analyte molecules are also frequently lost. Chromatographically, the matrix peaks can be separated from analyte peaks using GC x GC, where two columns with different stationary phases are used. Within the mass spectrometer, spectral resolution can be used, where unique ions for the target analyte are chosen that aren’t present in the coeluting matrix peaks. However, this frequently fails when analyzing these very complex samples, as fragment ions from the various coeluting matrix peaks have the same m/z as many ions from the target peak, skewing the ion ratios and resulting in false negatives or inaccurate quantitation.

Tandem mass spectrometry (MS/MS) uses multiple stages (mass analyzers) within the mass spectrometer to increase the sensitivity of the analyte by reducing the background from the coeluting matrix peaks. Different, chromatographically coeluting compounds may create molecular/fragment ions that have the same m/z; however, these ions will have a different structure. After ionization, the first mass analyzer selects the ion, called the precursor ion . The next stage is to fragment this further, which is usually achieved with an inert gas (e.g., argon) through a process called collision induced dissociation (CID) . The ions produced, called product ions , will be dependent on the structure of the precursor ion, therefore the product ion mass spectrum from the interferent(s) will be different from that of the target analyte. The next stage of MS/MS is then to separate the product ions using another mass analyzer before the ion detector. There are several different configurations of MS/MS instruments including a triple quadrupole (QqQ) where Q1 is used for precursor selection, q2 is used as the collision cell and Q3 is used as the product ion mass analyzer. Triple quadrupoles are the most common as they are unit mass instruments and therefore more affordable. An example of a triple quadrupole is shown in Figure 5 in multiple reaction monitoring (MRM) mode. Q-ToFs use a quadrupole for precursor ion selection and an HRMS ToF as the product ion mass analyzer. Ion traps are capable of performing MS n , with all stages occurring within the single trap. All ions are ejected except for the precursor ions and a voltage is applied that resonates with the energy to destabilize and fragment the precursor ion of that particular m/z. This process can then be repeated to fragment further, as many times as are needed.

Generally, MS/MS instruments are used for target analysis only and not for the routine analysis of unknowns. There are several modes of operation including MRM , single reaction monitoring (SRM) which is similar but more sensitive, product ion scans where Q3 is operated in scan mode to acquire the full product ion mass spectrum and neutral loss scans . MS/MS instruments can also be used as a standard MS—this is required during method development of MS/MS techniques—however, caution should be applied as the mass spectrum produced may be of a lower quality and affect the identification, particularly for scanning instruments. Some manufacturers also enable dual acquisition mode, where the acquisition alternates between e.g., MRM mode for target analysis and scan mode for the identification of unknowns.

Figure 5: An example of a triple quadrupole MS/MS instrument in multiple reaction monitoring (MRM) mode . Credit: Anthias Consulting.

Strengths and limitations of GC-MS

GC alone is limited in that it isn’t possible to identify unknown compounds using standard GC detectors, but this is possible when paired with MS. Conversely, direct analysis of samples using MS produces mixed mass spectra that can be difficult to deconvolute and interpret, especially when there are more than a few compounds in the sample. But pairing with GC gives the ability to separate the mixture.

Not all compounds can be separated on a standard GC column, therefore the ability to use spectral resolution to remove the interferences leads to more accurate quantitation. The deconvolution of mass spectra enables the detection of small peaks under baselines and matrix peaks, and selective MS/MS enables the identification of trace analytes in very complex samples 4 with little interference.

Isomers of an analyte usually have the same or very similar mass spectra and therefore it is very difficult to distinguish between them using just the mass spectrum. Therefore, chromatographic resolution is required to separate them. The individual isomers are identified by their retention times and good chromatographic resolution is required for accurate integration and quantitation. This is the same for homologous series, such as saturated hydrocarbons, where beyond a certain chain length, no molecular ion can be seen and the mass spectra all look the same. Again, the retention time must be used to determine the identity of the saturated hydrocarbon.

In terms of the lack of a molecular ion used in the identification of a compound, alternative softer ionization methods can be used. For example, EI at a lower eV or chemical ionization in positive or negative modes can result in fewer fragment ions and prevent the loss of the molecular ion or make its peak stronger. However, the fragmentation pattern is very much used for identification of the compound’s structure, and target libraries usually need to be created as few exist for soft ionization techniques, but this is improving as soft ionization techniques become more extensively used.

The hyphenation of GC and MS brings together two powerful techniques that are complementary to each other and that can be used for the separation and identification of compounds in a sample. When one method fails to provide answers, the other method can then be relied upon.

Common problems with GC-MS

The most common problems in GC-MS instruments are leaks into the system and system contamination .

The MS usually has a vacuum to reduce background interferences and ion-molecular reactions, to increase the lifetime of components, reduce maintenance and to avoid any electrical discharges, as high voltages are used within the MS. A very good vacuum is required to obtain good sensitivity and small leaks can increase the background whereas large leaks impede the vacuum system. High column flows increase the number of gas molecules entering the MS and the vacuum system cannot evacuate them fast enough to establish a good vacuum, so sensitivity reduces. Hence, when moving a method from GC to GC-MS, smaller internal diameter columns should be used with lower column flows. The maximum flow rate for high sensitivity analyses is dependent on the vacuum system of the mass spectrometer. This should be known and taken into consideration, along with the optimal flow rate of the carrier gas, for the best separation efficiency.

Mass spectrometers are sensitive instruments and regular maintenance is required to clean the ion source and replace the oil in the vacuum pump to retain good sensitivity. High molecular weight matrix from the sample should be retained in the GC inlet liner, by optimizing the inlet temperature, which is easily replaced rather than transferring into the column where it can damage the stationary phase and dirty the MS ion source. Another source of ion source contamination is column bleed . More stable “-MS” columns should be installed in a mass spectrometer and column conditioning should not be carried out when connected to the MS.

GC-related issues still occur, for example activity and sample degradation , especially where the sample has been through less sample preparation, for example if GC-MS/MS is used and the matrix can’t be “seen”.

Applications of GC-MS and GC-MS drug testing

GC-MS and GC-MS/MS are used in many industries for routine analysis looking for volatile contaminants with a molecular weight of usually less than 700 amu, for example in the food, 2 environmental, 5 forensics, 6 anti-doping 7 and consumer products 8 industries. GC-MS is also used in research to identify unknown volatile compounds, including in food and flavors, space, petrochemical, chemical, agriculture, tobacco, pharmaceutical, healthcare, energy, mining, environmental and forensics to name but a few.

Drug testing, as an example, can be carried out for multiple reasons: pathology, healthcare and anti-doping of both humans and animals. 9 Biological fluids like urine and blood samples are complex with large amounts of matrix and usually the drugs are at very low concentration. Therefore, their detection requires a hyphenated technique like GC-MS to start to separate the target or compounds of interest away from the matrix peaks. They can then be selectively identified using MS/MS and more frequently MS/MS with an HRMS and sometimes additional GC x GC separation.

1. Turner DC, Schäfer M, Lancaster S, Janmohamed I, Gachanja A, Creasey J. Gas Chromatography–Mass Spectrometry . Royal Society of Chemistry; 2020. ISBN-10:  1782629289

2. Hernández F, Cervera MI, Portolés T, Beltrán J, Pitarch E. The role of GC-MS/MS with triple quadrupole in pesticide residue analysis in food and the environment. Anal Methods . 2013;5(21):5875-5894. doi:  10.1039/C3AY41104D

3. Fialkov AB, Steiner U, Lehotay SJ, Amirav A. Sensitivity and noise in GC–MS: Achieving low limits of detection for difficult analytes. Int. J. Mass Spectrom. 2007;260(1):31-48. doi:  10.1016/j.ijms.2006.07.002

4. Bünning TH, Strehse JS, Hollmann AC, Bötticher T, Maser E. A toolbox for the determination of nitroaromatic explosives in marine water, sediment, and biota samples on femtogram levels by GC-MS/MS. Toxics . 2021;9(3):60. doi:  10.3390/toxics9030060

5. Ferrer I, Thurman EM. Advanced Techniques in Gas Chromatography-Mass Spectrometry (GC-MS-MS and GC-TOF-MS) for Environmental Chemistry: Volume 61 . Elsevier; 2013;2-502 ISBN:  9780444626240

6. Pasternak Z, Avissar YY, Ehila F, Grafit A. Automatic detection and classification of ignitable liquids from GC–MS data of casework samples in forensic fire-debris analysis. Forensic Chem . 2022;29:100419. doi:  10.1016/j.forc.2022.100419

7. Narciso J, Luz S, Bettencourt da Silva R. Assessment of the quality of doping substances identification in urine by GC/MS/MS. Anal Chem . 2019;91(10):6638-6644. doi:  10.1021/acs.analchem.9b00560

8. Karlberg AT, Albadr MH, Nilsson U. Tracing colophonium in consumer products. Contact Derm . 2021;85(6):671-678. doi:  10.1111/cod.13944

9. Clarke J, Rockwood AL, Kushnir MM. Mass spectrometry. In: Rifai N, Horvath AR, Wittwer CT, Hoofnagle A ed. Principles and Applications of Clinical Mass Spectrometry: Small Molecules, Peptides, and Pathogens . 1st ed.; Elsevier; 2018:33-65. ISBN:  9780128160633

About the author:

Diane is the senior consultant and director of Anthias Consulting Limited. She  has developed methods, given support and high-quality training for companies in most industries around the world for more than 20 years. A Warwick University Graduate, Diane completed her Masters in analytical chemistry and started her career in environmental chemistry, later gaining significant experience as an Applications Chemist. Diane's area of research through her PhD studies at The Open University was disease diagnosis. Diane is President-Elect of the Royal Society of Chemistry Analytical Division and Chair of the Analytical Chemistry Trust Fund. Diane is a visiting academic and consultant at The Open University.

What is gas chromatography mass spectrometry? Gas chromatography mass spectrometry (GC-MS) consists of two very different analytical techniques: gas chromatography (GC) which is hyphenated (hence uses a hyphen not a forward slash) to mass spectrometry (MS). Usually, the analytical instrument consists of a gas chromatograph that is hyphenated via a heated transfer line to the mass spectrometer, and the two techniques take place in series. However, some specialist and usually miniature or portable instruments contain the whole GC-MS within a single box.

• Open access
• Published: 16 April 2020

Recent developments and emerging trends of mass spectrometric methods in plant hormone analysis: a review

• Liyuan Wang 1 ,
• Yilin Zou 1 ,
• Han Yeong Kaw 1 ,
• Gang Wang 1 ,
• Huaze Sun 1 ,
• Long Cai 1 ,
• Chengyu Li 3 ,
• Long-Yue Meng 1 , 2 &
• Donghao Li 1  

Plant Methods volume  16 , Article number:  54 ( 2020 ) Cite this article

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Plant hormones are naturally occurring small molecule compounds which are present at trace amounts in plant. They play a pivotal role in the regulation of plant growth. The biological activity of plant hormones depends on their concentrations in the plant, thus, accurate determination of plant hormone is paramount. However, the complex plant matrix, wide polarity range and low concentration of plant hormones are the main hindrances to effective analyses of plant hormone even when state-of-the-art analytical techniques are employed. These factors substantially influence the accuracy of analytical results. So far, significant progress has been realized in the analysis of plant hormones, particularly in sample pretreatment techniques and mass spectrometric methods. This review describes the classic extraction and modern microextraction techniques used to analyze plant hormone. Advancements in solid phase microextraction (SPME) methods have been driven by the ever-increasing requirement for dynamic and in vivo identification of the spatial distribution of plant hormones in real-life plant samples, which would contribute greatly to the burgeoning field of plant hormone investigation. In this review, we describe advances in various aspects of mass spectrometry methods. Many fragmentation patterns are analyzed to provide the theoretical basis for the establishment of a mass spectral database for the analysis of plant hormones. We hope to provide a technical guide for further discovery of new plant hormones. More than 140 research studies on plant hormone published in the past decade are reviewed, with a particular emphasis on the recent advances in mass spectrometry and sample pretreatment techniques in the analysis of plant hormone. The potential progress for further research in plant hormones analysis is also highlighted.

Plant hormones are small organic molecules that naturally occurring in plants at very low concentrations. They regulate plants germination, growth, reproduction as well as both biotic and abiotic stress responses under different environmental conditions. These molecules show diverse chemical properties and unique chemical structures with wide polarity range and poor photo-thermal stability [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. Plant hormones are categorized into several classes based on their structures and physiological functions [ 10 , 13 ]. The most common classes are: auxin, cytokinin (CK), abscisic acid (ABA), gibberellin (GA), brassinosteroids (BR), salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and some newly identified plant hormones such as strigolactone (SL) [ 12 ]. Each class is defined by its physiological functions in regulating plant growth through synergistic or antagonistic [ 14 , 15 ]. Therefore, simultaneous quantification of the multi-class plant hormones and accurate determination of spatial–temporal distribution is important to clarify the mechanisms of their recognition, regulation, and how they influence plant growth and development.

Accurate determination of plant hormones requires the development of highly sensitive and efficient analytical techniques for analysis of plant hormone. Effective analysis of plant hormones is limited by inefficient extraction procedures due to their extremely low abundance (usually at ng g −1 or even pg g −1 level) and varied concentration ranges in plants [ 16 ]. For instance, the concentration of auxin and JA in plants ranges about 1–50 ng g −1 fresh weight (FW) whereas the content of BRs is as low as 0.01–0.1 ng g −1 FW [ 17 ]. This calls for highly sensitive analytical strategies with a good dynamic range for quantitative analysis are required for simultaneous extraction and purification of multi-class plant hormones. Existence of several structural isomers of plant hormones also complicates the separation processes. Another factor that affects analysis of plant hormone is the matrix effect. Elimination of interference is important for obtaining accurate information on the metabolism and functions of plant hormones [ 18 ]. In addition to these features, plant hormones are unstable and extremely sensitive to the environment change such as temperature, humidity and light due to their chemical heterogeneity. For instance, GAs show high sensitivity to pH and temperature (above 40 °C) but are relatively stable under acidic conditions [ 19 ].

These challenges necessitate the development of sample pretreatment and detection methods that are highly selective, sensitive, good enrichment, ease of handling and with broader dynamic range of detection. Non-chromatographic analytical methods such as bioassays and immunoassays were widely used in the early stage of plant hormone research. In comparison with mass spectrometry (MS)-based methodologies, these methods are less sensitive, specific and cannot perform simultaneous detection of multi-class plant hormones, thus, they have been phased out with modern approaches [ 20 ]. MS detection equipped with chromatographic techniques are the most widely used analytical approaches for simultaneous analysis of multi-class plant hormones are based on MS detection equipped with chromatographic techniques. Chromatographic methods integrated with MS system provide effective analysis for multi-class plant hormones in the plant matrix (plant component except for plant hormone) [ 20 , 21 , 22 , 23 ]. Particularly, combining gas chromatography (GC) or liquid chromatography (LC) with different MS detectors forms a rapid, sensitive and high-throughput method for quantitative analysis of plant hormone [ 21 , 24 , 25 , 26 ]. Furthermore, in order to simultaneous analysis multi-class plant hormones in a single step, a derivatization step is often required before MS-based instrumental analysis because of the non-volatile and non-ionizable property of some plant hormones [ 27 , 28 , 29 , 30 , 31 , 32 ]. Thus, derivatization step may promotes comprehensive investigation of the distribution of plant hormone among plant tissues, and hence characterization of metabolic and signaling processes involving plant hormones [ 33 ].

MS is a promising technique for efficient analysis of plant hormones. This is because it has high detection performance [ 1 , 11 , 33 , 34 , 35 , 36 ]. For this reason, MS-based methods with better sensitivity, selectivity, accuracy and high-throughput features are used to quantitatively and qualitatively analyze plant hormone at tissue and cellular level [ 29 , 37 , 38 , 39 ]. Given its effective mass-to-charge (m/z) separation ability, mass analyzer provides enhanced selectivity of analytes, thereby improving quantification of trace and ultra-trace plant hormones in plant. Several types of mass analyzers have been developed such as ion trap (IT) [ 40 ], quadrupole time-of-flight (Q-TOF) [ 41 ] and triple quadrupole instruments (QQQ) [ 42 ]. Mass analyzer combined with chromatography have been extensively applied in plant hormone analysis, indicating their ability of accurate quantitative analysis and efficient separation capabilities [ 43 ]. MS-based methods can facilitate simultaneous analysis of multiple target compounds. It is therefore suitable for comprehensive analysis and elucidation of the biosynthesis, transportation, metabolic pathways, and signaling networks of plant hormones.

This review provides an up-to-date overview of modern analytical methods and sample pretreatment techniques for plant hormone analysis. Some recent applications of these methods are highlighted in this review. The fragmentation patterns of plant hormones are investigated to provide a theoretical basis for the establishment of a mass spectral database. The prospects related to the analysis of plant hormone are also proposed, which would serve as a technical guide for further discovery of new plant hormones [ 44 , 45 ].

• Sample pretreatment

The key sample pretreatment strategies applied in plant hormone analysis include homogenization, extraction and purification steps (some studies incorporate a derivatization or labeling step depending on the analytical strategies and instrumental requirements) [ 42 , 43 , 46 , 47 , 48 , 49 , 50 ]. Sample pretreatment procedure is the major bottleneck that limits rapid separation and simultaneous analysis of multi-class plant hormones, especially at trace levels. To effectively extract plant hormone from complex plant samples, analytical methods that minimize matrix interferences and meet the requirements of high sensitivity, satisfactory recovery and good reproducibility with simple operation are desirable [ 1 , 19 , 23 , 36 , 40 , 49 , 50 , 51 , 52 ]. Previous studies have indicated that sample pretreatment is the most labor-intensive and time-consuming step in the whole analytical procedure of endogenous plant hormones [ 43 ]. In addition, sample pretreatment is the most error-prone part of the process accounting for about 30% of all sources of errors [ 10 , 13 , 18 , 43 ]. An ideal sample pretreatment technique should be less sample consuming, exhibit high sensitivity, and display outstanding enrichment capabilities, especially during in vivo detection.

Accurate determination of variations in the spatial distributions of plant hormone with the aspects of in vivo analysis them in a trace amount of plant tissues would broadening the scope of investigating their physiological functions. The extremely low concentration of plant hormone, similarity in chemical structures and large amount of metabolites in plants are the main challenges which affect accurate detection of plant hormone as they cause interference during analyses [ 53 ]. A trace amount of sample is sufficient for investigating the content and dynamic changes of plant hormones in a specific part of the plant. This limits the use of rare and endangered plants as source of sample for analytical purposes. However, only few pretreatment methods are sensitive enough and provide high enrichment capabilities for in vivo analysis of trace amount of plant hormones in plant matrix [ 1 , 51 , 53 , 54 , 55 ]. On-line analysis methods provide a platform for analysis of trace compounds analysis [ 56 ]. However, due to the complexity of plant sample matrix and the low concentration of components to be measured, development of on-line techniques has been relatively very slow. In addition to in vivo, in situ, real-time and on-line analysis, sample pretreatment techniques are expected to be highly sensitive with good enrichment properties to produce reliable analytical results for plant samples with high complexity. Typical examples of sample pretreatment methods published in the recent past 5 years are shown in Table  1 .

Common pretreatment methods in plant hormone analysis

Depending on different extraction modes, extraction method can be divided into classic extraction methods and microextraction methods. Both classic extraction and microextraction methods are based on liquid phase extraction and solid phase extraction [ 62 ]. Classic extraction methods provide near-exhaustive extraction efficiency [ 45 ]. Compared with classic extraction methods, microextraction methods have the advantages of having high extraction efficiency and miniaturization of device and enrichment capacity making them more suitable for the analysis of plant hormone [ 47 ]. The extraction and clean-up techniques for trace compounds analysis include liquid–liquid extraction (LLE) [ 17 , 21 , 24 , 30 , 63 , 64 , 65 ], liquid phase microextraction (LPME) [ 52 , 56 , 66 ], dispersive liquid–liquid microextraction (DLLME) [ 40 , 67 ], polymer monolith microextraction (PMME) [ 68 ], solid phase extraction (SPE) [ 26 , 38 , 55 , 69 , 70 , 71 , 72 ], solid phase microextraction (SPME) [ 25 , 34 ], magnetic solid phase extraction (MSPE) [ 16 , 23 , 73 ], matrix solid-phase dispersion (MSPD) [ 37 ], dispersive micro solid phase extraction (DMSPE) [ 74 ], ion pair stir bar sorptive extraction (SBSE) [ 1 ] and electromembrane extraction (EME) [ 75 ] (see Fig.  1 ).

Sample pretreatment methods for the determination of plant hormone. Sample pretreatment methods: LLE liquid–liquid extraction, LPME liquid phase microextraction, DLLME dispersive liquid–liquid microextraction, SPE solid phase extraction, SPME solid phase microextraction, MSPE magnetic solid phase extraction, MSPD matrix solid-phase dispersion, SDSE single drop microextraction, SBSE stir bar sorptive extraction, DMSPE dispersive micro solid phase extraction

The conventional LLE and SPE methods are widely used to analyze biological samples [ 76 ]. However, these sample pretreatment techniques are associated with drawbacks such as complicated operations, time-consuming, the requirement of relatively large amounts of sample and organic solvents, strong matrix effect (the interference and influence of matrix on analyte analysis) and the difficulty to automate. Flokova et al. [ 55 ] achieved rapid extraction of plant hormone with less sample consumption, less than 20 mg FW of A. thaliana leaf tissue in 10% methanol. They used single-step reversed-phase polymer-based SPE method which reduced the matrix effect and increased recovery of labile plant hormone.

Recent technological advancements have improved the miniaturization and automation of sample pretreatment methods. Compared with the traditional LLE and SPE methods, microextraction techniques such as DLLME, LPME, SPME and DMSPE applications in trace analysis are ever increasing. The solvent consumption, better extraction performance, minimized invasiveness and automated coupling with analytical instruments property of microextraction techniques have evidently benefited (Fig.  1 ) [ 62 ]. Cai et al. [ 77 ] developed a clean-up strategy employing a single-step dispersive solid-phase extraction (DSPE) combined with UPLC–MS/MS to obtain spatial–temporal information of 54 plant hormones including auxins, ABA, SA, JA, GAs and CKs. Wang et al. [ 75 ] used a combination of EME and LC–MS/MS to quantitatively detect six acidic plant hormones in 20 mg citrus leaf sample with a limit of detection (LOD) ranging from 0.1 to 10 ng mL −1 . For more accuracy sample processing, sample pretreatment techniques require automation. Wang et al. [ 37 ] used an in-line coupled MSPD-MAX-MCX system to analyze a wide range of polar plant hormones in rice (200 mg). The proposed method showed higher extraction efficiency, lower matrix effect, ease of manipulation and time-saving characteristics. Recently, off-line procedures with highly efficient separation and detection sensitivity have been used to analyze plant hormone. However, the possible loss of trace plant hormone during additional process (compare with in-line procedures) has been an intractable challenge. Direct on-line analysis (coupling of sample preparation techniques directly to the separation and detection system) minimizes sample preparation steps and enables the effective pre-concentration and clean-up of plant samples [ 78 ]. Although these microextraction methods achieve effective plant hormone extraction, some of them are difficult to automate for on-line analysis.

SPME strategies and advances

An ideal sample pretreatment technique for plant hormones analysis should be simple, rapid, selective, efficient, solvent-free, inexpensive, reproducible, highly accurate and avoids the degradation of analyses. For simultaneous quantitative analysis of multi-class plant hormones, the compatibility of an in vivo and automated sample pretreatment methods with dynamic and ultra-sensitive extraction capacity are required. SPME has advantages in these aspects [ 48 , 50 , 52 , 79 ]. In SPME, sampling, extraction, concentration, and injection are integrated into a single step, which promotes miniaturization, automation, in vivo sampling and on-line analysis [ 80 ]. However, commercialized SPME sorbents exhibit lack selectivity which affects the distribution between analytes and the stationary phase. To address this challenge, several modified SPME sorbents have been designed to improve the sensitivity and selectivity of the extraction process [ 81 ]. Liu et al. [ 82 ] used a carbowax-coated fiber as SPME sorbent hyphenated with HPLC to detect auxin. They found that the modified sorbent had a relatively much higher extraction efficiency compared to polyacrylate fibers. Its dynamic range spanned over three orders of magnitude. The LOD/limit of quantification (LOQ) values of the target compounds in pure water were 0.149 (0.497), 0.442 (1.472), 0.121 (0.403) and 0.058 (0.193) μg L −1 for IAA, ABA, IBA and NAA, respectively. Song et al. [ 61 ] applied the SPME method incorporating on-cyclodextrin (–CD)-modified carbon nanotubes (CNTs) and a hollow fiber (HF) to analyze 1-naphthaleneacetic acid (NAA) and 2-naphthoxyacetic acid (2-NOA) in vegetables. This strategy allowed easy handling and provided environmentally friendly features as it minimized the amount of solvents. In most cases, plant hormones in plant tissues are determined simultaneously by combining SPME and HPLC [ 62 , 76 , 77 ,, 78 ]. Wang et al. [ 34 ] developed an automated and sensitive method to analyze the endogenous 28-epihomobrassinolide (28-epihomoBR) in A. thaliana by coupling on-line SPME with a polymer monolithic column and LC–MS (SPME-LC–MS). The poly (methacrylic acid- co -ethylene dimethacrylate) (poly (MAA- co -EDMA)) monolith was prepared in the capillary. The proposed SPME had satisfactory recovery (80.3–92.1%) and reproducibility (RSD 6.8–9.6%). Thus, SPME notably reduces assay duration and the difficulty of automation and improves in vivo analysis.

The application of SPME for in vivo analysis not only simplifies the extraction process, but also has the advantages of non-destructive extraction and easy to couple with mass spectrometry. Thus, this method facilitates on-site sampling, pretreatment and the detection of unstable analytes, making it suitable for long-term monitoring of signaling processes of plant hormones.

Mass spectrometric analysis

Co-extraction of lipids and other interfering compounds is often eliminated by applying enrichment and purification methods. However simultaneous and accurate determination of individual or multi-class plant hormones in plant samples is still a great challenge due to the complex plant matrix [ 26 , 80 , 81 , 83 ]. Various strategies including biological approaches (e.g. bioassays and immunoassays) physical and chemical analytical methods have been proposed to overcome the matrix effect of plant samples during the quantification of plant hormones [ 18 ]. Despite the high sensitivity of these analytical methods, most of them are not effective enough for the simultaneous analysis of multi-class plant hormones. MS-based technologies overcome these limitations. Successful exploitation of MS-based technologies for determining plant hormone from plant tissue satisfy the needs of high sensitivity. Furthermore, MS-based technologies can be combined with different chromatographic methods, which make it possible for high throughput analysis of plant samples [ 32 , 55 , 61 , 75 ].

Plant hormone in plants were analyzed by GC–MS initially, in which the separation was dependent on using different partition coefficients between the gas phase and the stationary phase [ 20 , 26 , 56 , 84 , 85 , 86 ]. However, most plant hormones are non-volatile, therefore a derivatization step is integral, not only it converts the non-volatile plant hormones to more volatile and thermally stable derivatives, but also for changing chromatographic behavior, improving detection sensitivity and selectivity of the analytes [ 87 ]. Based on the non-volatility property of most plant hormones, liquid chromatography has high separation performance when analyzing non-volatile compounds without the necessity of derivatization, which reduces possible loss of the target compound during derivatization procedure.

LC–MS analysis

LC is by far the most common analytical technique used to qualitatively and quantitatively analyze trace plant hormone due to its high separation performance, high detection sensitivity, fast analytical speed and easy operation [ 60 ]. LC hyphenated with MS detection provides higher sensitivity and accuracy for the simultaneous analysis of multi-class plant hormone, which promotes research on the molecular biology of plant hormones [ 88 ]. Different LC–MS techniques used to analyze plant hormone are shown in Table  2 .

The ion source allows large, non-volatile molecules to be analyzed directly from the liquid phase, which ionizes the neutral atom or molecule and fragment the ion beam generated from it [ 93 ]. Two of the frequently used ion sources in plant hormone analysis are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). ESI is preferred by most scientists as it has well balanced ionization efficiency for various classes of chemical compounds and is a soft ionization method [ 94 ]. It is, therefore, suitable for analysis of polar compounds because it does not require the rapid vaporization of solvents and ions can be generated in solution [ 95 ]. Since most plant hormones are polar in nature, ESI is comparatively a more suitable ion source for the characterization, identification and quantification of plant hormone in plant tissue [ 96 ].

Ionization efficiency of plant hormone analysis

The effectiveness of ionization method greatly influences the sensitivity of LC–MS analysis. Factors affecting the efficiency of ESI process includes composition and flow rate of mobile phase as well as matrix complexity of the plant sample [ 97 ]. The purification step of plant sample is crucial to improving the ionization efficiency, accuracy and sensitivity of LC–ESI–MS/MS analysis as it removes interfering co-eluting compounds which cause ion-suppression when using ESI, hence, affecting the ionization efficiency of plant hormone [ 98 ]. It is also important to optimize the composition, type and concentration of mobile phase and the purity of sample to ensure effective ionization during LC–ESI–MS analysis [ 99 , 100 ]. Na + , K + and NH 4 + are commonly present in the solvent throughout the transportation or storage processes, which are classical additives and buffers of LC. Some mobile phase additives such as formic acid, acetic acid, trifluoroacetic acid are usually used to improve the ionization efficiency during MS detection. The chemical properties, concentration, and pH of the mobile phase additive have a significant effect on the LC–MS response [ 60 , 87 ]. The type of appropriate additive added in mobile phase is critical to improve the chromatographic resolution, peak shape and ionization efficiency. Formic acid or acetic acid are the commonly used additives in the mobile phase to detect basic plant hormones in positive ion mode, whereas ammonium formate is used as the additive of the mobile phase to detect the acidic plant hormones in negative ion mode [ 101 ].

ESI is an ionization technique that converts ions in solution to gas-phase for MS analysis. The first step of ESI is the electrophoretic migration of anion and cation in the solution under an electric field to produce ions [ 102 ]. The efficiency of ion production depends on conductivity, which relies on the concentration of electrolyte. Suitable solvents for ESI include polar and moderately polar solvents and the most frequently used solvents are methanol, acetonitrile and water. Composition of the mobile phase for plant hormone analysis is usually water and acetonitrile (ACN) or methanol with suitable additives [ 103 ]. Methanol is an appropriate solvent for ESI–MS analysis of acidic compounds in the negative ionization mode. The response of some analytes in negative ESI–MS can be improved by applying methanol. In contrast, ACN is more suitable for analyzing in positive ion mode as it is an aprotic solvent [ 103 ]. The flow rate of mobile phase in the ESI ion source also influences the ionization process. Normally, smaller initial droplet is observed in the lower flow rate which decreases the number of fission cycles and the amount of solvents to be evaporated to form gas-phase ions, thus improving the electrospray effect [ 104 ].

Given the above-mentioned factors that affect the ionization efficiency, where the composition of the mobile phase affects the LC procedure. The LC analysis of plant hormone is often performed by combining water and methanol or acetonitrile with the addition of formic acid or ammonium formate as the mobile phase. The flow rate of mobile phase is often optimized at 0.2–0.5 mL min −1 . Cai et al. [ 73 ] successfully separated and analyzed 8 CKs, IAA, ABA, JA and 10 GAs using a 27-min gradient of 0.05% FA H 2 O (A) and ACN (B) at a flow rate of 0.4 mL min −1 . Suh et al. [ 75 ] characterized acidic plant hormones (JA, ABA, SA and GAs) in plant tissues using a linear gradient profile of 0.1% formic acid in water and (A) 0.1% formic acid in methanol (B) at 0.3 mL min −1 .

Selection of mass analyzers for plant hormone analysis

Accurate identification of the mass-to-charge ratio of the ion peak is the principle of mass spectrometry and quantification. High resolution enhances the sensitivity of quantitative analysis [ 105 ]. One of the mainstream industries where LC–MS is constantly implemented is the doping control analysis, which employs different mass analyzers such as triple quadrupole (QqQ) [ 106 ], time of flight (TOF) [ 107 ] and quadrupole time of flight (QTOF) [ 108 ]. These qualitative and quantitative approaches are also applied in the detection of plant hormones. The use of multiple QqQ mass spectrometers to form a tandem mass spectrometry (MS/MS) not only enhances the qualitative capability of the mass spectrometer, but also retains the original quantitative capability of the quadrupole mass spectrometer. Several modes including full scan mode (Scan mode), selected ion monitor mode (SIM mode), multiple reaction monitor mode (MRM mode) and selected reaction monitor mode (SRM mode) are available in QqQ mass spectrometry [ 109 ]. Compared with the full scan mode in which all ions are simultaneously monitored, MRM mode provides higher selectivity and specificity to analyze multiple plant hormones. Fragmentation patterns of multi-class plant hormones in ESI–MS MRM mode (ET in Scan mode) are shown in Fig.  2 . The conditions for MRM transition of plant hormones are listed in Table  3 . Another mass analyzer widely used to detect plant hormones is TOF–MS [ 92 ]. TOF–MS exhibits faster detection speed, higher resolution and broader dynamic range of detection compared to QqQ-MS. The combination of TOF–MS and QqQ-MS provides broader application prospects (quadrupole time-of-flight mass spectrometer (QTOF-MS)) [ 98 , 99 , 110 ]. The quantitation of plant hormone by mass spectrometry is important for discovering the potential plant hormones in plants. Yang et al. [ 111 ] proposed identification of ripening degrees and cultivation regions of mulberries analysis, which employed high detection sensitivity and accuracy. Thus, LC–MS techniques provide an easy and robust method for plant hormone analysis.

Fragmentation ions of multi-class plant hormones by triple quadrupole mass spectrometer (except ET)

Chemical labeling

Over the past decades, the trend of plant hormone analysis has lied in a tiny amount sample consumption with sensitive performance [ 35 , 115 , 116 ]. However, most of the plant hormones were present in plant at ultra-low levels, while many classed of them exhibit with low ionization efficiency in MS [ 16 , 40 ]. Benefit from high resolution of MS, chemical labeling may serve as a strong tool to enhance sensitivity and accuracy of determining plant hormones [ 11 , 26 ], which overcome the problem by synthesizing series of stable isotope-coded reagents for chemical derivatization to the target compounds during sample pretreatment or MS detection procedure [ 117 ]. The stable isotope labeling affects the accuracy of the sample pretreatment procedure and apply the possibility to directly analyze plant hormones in the extract with relative reliability [ 118 , 119 ]. When combine the light and heavy isotope labeled control and sample groups, the error that introduced during sample pretreatment step would compensate and the reliable relative quantification results then obtained [ 120 ]. Li et al. [ 112 ] were simultaneous relative quantification of 8 acidic plant hormones in sub-milligram amount of plant materials by stable isotope labeling by bromocholine bromide (BETA) and its deuterated counterpart D 9 -BETA, which improved sensitivity of 1–3 orders of magnitude. Furthermore, to improve the sensitivity of MS detections, amines are applied for the labelling (derivatization) procedure to form amides by making plant hormones easily ionized [ 31 , 121 ]. Deng et al. [ 115 ] discovered a quaternary ammonium derivatization reagent (4-borono- N , N , N -trimethylbenzenaminium iodide) for ultrasensitive analysis of five BRs with vicinal diol functional groups on the side chain. The ionization efficiencies of the BRs enhanced by 1190–448,785 times and the minimal detectable amounts (MDA) of five target BRs were decreased to attomolar levels. The stable isotope labeling can improve the sensitivity and reproducibility of the plant hormone analytical procedures [ 19 , 26 , 112 ], which provide applicable capability in analyzing the plant hormone distribution and transportation in different plants.

Mass spectrometric analysis of plant hormone

Acidic plant hormones, auxin, abscisic acid (aba), jasmonic acid (ja) and salicylic acid (sa).

Typical acidic plant hormones comprise majority of the auxin, abscisic acid, jasmonic acid and salicylic acid [ 101 , 102 ], which exhibit good response in MS negative ion mode. The quantitative analysis of these plant hormones in A. thaliana , wheat, Aloe , etc. have been realized by LC–MS/MS. Specific precursor-product ion pairs for each compound were monitored in MRM mode under the optimized fragmentor and collision energies [ 38 , 55 , 63 ]. A neutral CO 2 molecule is easily lost during the ionization of plant hormone containing carboxyl group while [M-H-CO 2 ] − fragment ions are produced in the negative ion mode [ 54 ]. ABA only shows good ionization efficiency in the negative ion mode and releases [M-H] − as a molecular ion peak [ 17 ]. JA liberates [M-H-COOH-C 5 H 9 ] − to form MW 58.7 product ion [ 55 ]. [M-H-R] − fragment ions are produce from SA in negative ion mode [ 122 ] (Fig.  2 ). Specific precursor and product ions for each class of plant hormone were identified by distinctive fragmentation patterns to achieve accurately qualitative and quantitative analysis of multi-class plant hormones.

Xiao et al. [ 35 ] used a sensitive LC–MS method based on N -(acridin-9-yl)-2-bromoacetamide (AYBA) to examine the interactions between ABA, IAA and JA in germinated rice seeds. Good precisions with RSD 1.5–13.8% (intra-day) and 1.2–7.3% (inter-day) and acceptable recoveries (88.6–102.9%, n = 6) were achieved through this method. Luo et al. [ 16 ] developed a method for analyzing plant hormones including IAA, ABA, JA, GAs, CKs and BRs in 100 mg (FW) of Brassica napus L. The samples were purified and separated by sequential magnetic solid-phase extraction (MSPE) and then analyzed by an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) with the LOD (S/N = 3) ranging from 0.45 to 126.1 pg mL −1 . The amount of plant tissues samples required has been reduced significantly from g (FW) to mg (FW). Cao et al. [ 70 ] developed a high-throughput method using liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ-MS) for the profiling and quantification of 43 plant hormones and their major metabolites including auxins, ABA, JA, SA, CKs and GAs in a single sample extract. A binary solid-phase extraction with polymer anion and polymer cation exchange resins has been used as a sample pretreatment method to obtain LOD ranging from 0.03 to 29.7 fmol. The higher sensitivity of this method has enabled the detection of plant hormones at low concentration. Wu et al. [ 25 ] used in vivo SPME to detect GAs, JA and ABA plant hormones in 20 mg (FW) of aloe leaf with a spatial resolution of 3–8 mm 3 and obtained an LOD of 60 pg g −1 .

Gibberellin (GA)

Being a class of tetracyclic diterpenoid carboxylic acids with either ent-gibberellane or 20-nor-ent-gibberellane carbon skeletons, GAs comprise of at least 136 highly similar endogenous analogs [ 123 ]. GAs appear only at ultra-trace level in plants ranging from fmol to pmol per gram (FW). Apart from their poor detect-ability, the complexity and the similar chemical structures of ultra-trace GAs pose a greater analytical challenge [ 118 ]. LC-MS/MS is the most widely used method for simultaneous determination of multiple GAs, however their ESI–MS analysis has poor sensitivity because of low ionization efficiency in the negative ion mode [ 124 ]. Since tetracyclic diterpenoids are stable molecules, they are easily detected by mass spectrometry as a result of fragmentation through the cleavage between functional groups. MS/MS study of GAs is usually carried out in the negative ion mode because they do not form [M+H] + in positive ion mode. Further, in the MS/MS analysis, the secondary fragmentation of GA 3 in negative ion mode [M-H] − produces [M-H-CO 2 ] − (m/z 301), [M-H-CO 2 -CO 2 ] − (m/z 257) and [M-H-CO 2 -CO 2 -H 2 O] − (m/z 239) [ 125 ]. Under different collision energies, the high abundance of m/z 143 ion is observed in the MS 2 of MS/MS spectrum indicating a stable product ion of GA 3 [ 126 ]. Based on the fragmentation pathway, m/z 143 is generated by m/z 239 ions with the loss of [M-H-CO 2 -CO 2 -H 2 O-H 2 O-C 6 C 6 ] − (Fig.  2 ). Despite the clear fragmentation pattern of GAs, the detection of GAs is still challenging due to its extremely low content [ 21 ].

Chemical-label based LC–ESI–MS/MS is commonly used for analyze GAs by introducing positively charged moieties to improve the ionization efficiency and detection sensitivity. For example, derivatization reagents with quaternary and tertiary amine group were employed for GAs labeling to enhance the ionization efficiency (the derivatization methods are mentioned in Table  4 ) [ 121 , 127 ]. These analytical methods are beneficial for elucidating the biosynthesis and distribution of GAs in plants.

The derivatization reaction is relatively difficult since it should be carried out in mild conditions because the structure of GAs undergoes different rearrangement reactions under varying pH. For example, GA 3 and GA 7 undergo lactone rearrangement under alkaline conditions (pH > 8.0) while a structural rearrangement of the C/D rings and hydration of the 16,17-double bond occurs in GA 1 under strong acidic conditions (pH < 1.0). Bromocholinebromide (BETA) and its deuterated counterpart D 9 -BETA have been used to derivatize acidic plant hormones including GAs from sample extracts. ESI signal enhancements of 1–3 orders of magnitude were achieved under optimized conditions but a derivatization temperature of up to 95 °C was required [ 112 ]. 3-bromoactonyltrimethylammonium bromide (BTA) is used to increase the ionization efficiency of GAs. In a previous study, BTA was added prior to detection for nano-LC-ESI-Q-TOF–MS analyze GAs and LODs ranging from 1.05 to 122.4 pg mL −1 were obtained [ 130 ]. Li et al. [ 19 ] introduced a one-reagent labeling technique by using only N -(3-dimethylaminopropyl)- N -ethylcarbodiimide hydrochloride (EDC) to directly react with the ultra-trace GAs. This innovative labeling reaction was performed at pH 4.5 and 35 °C to meet the optimal conditions of pH 2.5–8.5 and a temperature of below 40 °C for analyze GAs. Protonated EDC reacts with the carboxyl group of GA to form an unstable O-acylurea, which subsequently rearranges to a stable and amine-irreplaceable N-acylurea product through O/N migration. The sensitivity is increased by 500- to 1000-fold as compared to unlabeled GAs, furthermore, this method can directly label GAs down to about 0.1 pM as it eliminates extra reagents.

Basic plant hormones

Cytokinin (ck).

At present, the number of identified CKs exceeds 40, of which nucleosides (ribosides), nucleotides, and glycosides (O and N-glycosides) are free bases. The most naturally occurring CKs are N 6 -substituted adenine derivatives with an isoprenoid or aromatic side chain [ 106 , 107 ]. The mass spectrometry studies of CKs is usually carried out in positive ion mode, since they are easily protonated ([M+H] + ) [ 131 ]. After fragmentation, the loss of nucleosyl, glycosyl, or nucleotidyl group and the fragmentation of the N 6 -substituent for aglycons are observed in the spectrum. The representative plant hormone for CKs is trans -zeatin ( t Z) [ 90 ]. When a precursor ion is formed, the N–H bond on the t Z side chain cleaves to form a m/z 136 product ion (C 5 H 4 N 5 ) [ 132 ].

Antoniadi et al. [ 39 ] applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with in-tip micro-SPE and an ultra-high-sensitive MS technique to analyze the biosynthesis and homeostasis of CK at cellular level, which revealed a LOD of 0.01–1.0 fmol. Different LC systems can influence the sensitivity of MS. For instance, a sensitive assay of cytokinins was developed using PMME/HILIC/ESI–MS/MS which enhanced the MS sensitivity for cytokinins by threefold using HILIC as compared to the use of conventional reversed phase liquid chromatography (RPLC) with a mobile phase of 85% acetonitrile with 0.01% (v/v) formic acid and 15% water with 0.01% (v/v) formic acid. The LODs for the targets ranged from 0.0028 to 0.068 ng mL −1 , and the intra-day and inter-day RSD of this method were less than 12.7% [ 68 ]. The choice of liquid chromatography system mainly depends on the enrichment ability of sample pretreatment and the sensitivity of mass spectrometry. Scott et al. [ 133 ] developed a conventional HPLC system that demonstrated good sensitivity without the need for an ultra-high performance liquid chromatography-mass spectrometry (UHPLC–MS) system. This method was used to screen 17 CKs (IAA, ABA and AMP), Ado and Ino within 15 min. The rapid analytical and high-throughput capabilities of this method enabled the analysis of close to 100 samples per day, with LOD ranging from 2 pM for (9G)Z to almost 750 pM for indole-3-acetic acid. This method is well suited for functional genomics platforms tailored for understanding CK metabolism.

Neutral plant hormones

Brassinosteroid (br).

All BRs contain a four-ring 5α-cholestan skeleton connected to a side chain. The diversity of BR structures is caused by variations in the A and B rings as well as the substituent groups of the side chains. BRs play a crucial role in many plant developmental processes although present in trace levels (down to 0.01–0.1 ng g −1 FW level) [ 92 , 134 , 135 , 136 , 137 , 138 ]. Therefore, ideal sample pretreatment techniques should exhibit ultra-high sensitivity. Among the currently known BRs, brassinolide (BL) show the strongest activity, for BL fragmentation pattern, although the orientation of spatial position of the –OH in BL varies, ions generated from BL in the tandem mass spectrometry are substantially identical [ 139 ]. When BL fragmentation, as it shown in Fig.  2 , the loss of water molecules and other neutral fragments are observed in the spectrum (BL lost two molecules of water to form the m/z 459 ion).

The commonly used detection methods such as MS are not suitable enough for BRs detection due to neutral nature with no ionizable group and the stronger hydrophobicity of BRs than other plant hormones [ 118 , 124 , 125 , 126 ]. Therefore, the analysis of BRs requires highly selective sample preparation and highly sensitive detection with LC–MS after derivatization [ 29 , 36 , 40 , 121 , 126 , 140 ]. Lv et al. [ 32 ] developed a novel hyphenated approach based on ultrasonic-assisted dispersive liquid–liquid microextraction (UA DLLME) after derivatization (using 9-phenanthreneboronic acid) for the determination of BL and obtained LOD of 8.0 ng L −1 . Huo et al. [ 31 ] proposed a new labeling reagent, 2-bromopyridine-5-boronicacid (BPBA) for derivation brassinosteroids. This is a very simple and rapid labeling procedure that remarkably increases the sensitivity of BRs detection and LOD for the three BRs of 2.0 to 8.0 ng L −1 . The automated extraction of BRs can be performed by an on-line SPME with polymer monolith coupled to LC–MS for the analysis of endogenous 28-epihomobrassinolide (28-epihomoBR) in A. thaliana . The BR was derivatized with 3-(trimethoxysilyl) propylmethacrylate prior to detection which revealed a highly sensitive result with LOD of 2.0 ng L −1 [ 34 ]. The combination of several purification methods provides an efficient and sensitive processing of plant samples. Recently, Wang et al. [ 37 ] quantified six endogenous BRs with good compatibility and sensitivity by eliminating non-polar, polar and ionizable interferences using a matrix in-line MSPD–MAX–MCX coupled with HPLC–MS/MS to obtain LODs ranging from 0.008 to 0.04 ng mL −1 . In another study, in situ derivatization (ISD) was introduced and coupled with multiple sample preparation methods to simplify the process of extraction and derivatization [ 141 ]. Luo et al. [ 42 ] developed a solid phase boronate affinity labeling (SPBAL) and extraction technique, followed by a desorption & salt-induced phase transition extraction (SPTE) for further purification to rapidly determine endogenous BRs in plant tissues. The addition of a boronate affinity labeling reagent (4-PAMBA) for ISD lead to a 923–15,000-fold increase in sensitivity of BRs detection, with LODs ranging between 1.4 and 2.8 pg mL −1 .

Strigolactone (SL)

Strigolactones (SLs) have been suggested to act as a long-distance branching factor that suppresses the growth of preformed axillary shoot buds. They are derived from carotenoids and were recently recognized as a new family of plant hormone [ 59 , 91 , 111 , 142 ]. Typically, the natural SLs identified so far consist of tetracyclic skeleton (A, B, C, and D rings) with a tricyclic lactone (ABC rings) connected by an enol ether group to an α,β-unsaturated furanone moiety (D ring) [ 114 ]. SLs show relatively lower ionization efficiency because of their neutral structure which is similar to BRs which cause interference during MS detection. The lactone structure of C and D rings in SL molecules exhibits lower stability as they can hydrolyze in acidic or alkaline conditions leading to a more complicated sample preparation and instrumental analysis [ 44 ]. Except for the same structure of D ring, some of the ABC rings in SLs contain –OH, –CH 3 , –COOH, –CH 2 CO and –O– groups. The compounds with these functional groups including D ring can easily be cleaved to form fragment ions during the cleavage process in MS detection. SLs are usually detected in the positive ion mode. A typical SLs-GR24 (C 17 H 14 O 5 ) has a molecular weight of 298, and the main fragment ions formed are m/z = 97 and m/z = 185 [ 44 , 114 , 143 , 144 ]. The typical product ion of SLs (m/z = 97) is mainly the enol ether found between the ABC ring and the D ring in the GR 24 structure [ 144 ].

Yoneyama et al. [ 114 ] analyzed SLs in root exudates from 12 Fabaceae plants employing LC–MS/MS to clarify the regulation of SL production and revealed that exudation is closely related to the nutrient acquisition strategy of plants. Xie et al. [ 44 ] also used LC–MS/MS to characterize five different stimulants including four SLs from the root exudates of tobacco providing a valuable analytical strategy for determination of SLs. Foo et al. [ 143 ] used UPLC-MS/MS to quantify SL levels in the root exudate of 30-day-old rms1-2T and wild-type plants using deuterium-labeled internal standards. This was the first direct evidence that shooting does not majorly contribute to the SL levels in roots. Kohlen et al. [ 144 ] performed SL detection using HPLC -MS/MS, and the MRM transitions of [M+H/Na] +  > [M+H/Na-D ring] + and [M+H/Na] +  > [D ring] + were selected. Two SLs (orobanchol and orobanchyl acetate) were identified in Arabidopsis and the presence of a third (5-deoxystrigol) SL was discovered. These results show that xylem-transported SLs contribute to the regulation of shoot architectural response to phosphate-limiting conditions.

Conclusions

Major technological advancements in sample pretreatment methods and mass spectrometric methods in recent years has substantially facilitated plant hormone analysis [ 117 , 145 , 146 , 147 , 148 ]. To date, MS studies have identified many plant hormones and their metabolites that might be involved in the molecular mechanisms and physiological functions of plant development, but the analysis at unicellular level has remained a challenge. Several large-scale studies on plant hormone have been reported, these studies have promoted our understanding on the dynamic spatial–temporal distribution of plant hormone [ 19 , 35 , 77 ]. Current sample pretreatment methods for plant hormone is expected to be further optimized for realizing the in situ, real-time and high spatial resolution. The frontier research of analytical methodology will broaden the development of sample pretreatment techniques and derivatization strategies for more accurate characterization of plant hormone.

Going forward, the existing methods need to be improved to enhance the efficiency of sample treatment and chromatographic-mass spectrometry analysis of plant hormone in the following aspects:

Sample pretreatment methods with the ability to achieve real-time in situ and in vivo analysis of the spatial–temporal distribution of plant hormone are required to facilitate further studies on plant hormone regarding the synthesis, metabolism, transportation pathway and functional effects. Therefore, it is anticipated that the development of highly efficient sample pretreatment methods for in vivo and real-time analysis. The efficiency methods are likely to dominate future research to improve the characterization of the distribution of multi-class plant hormones within a whole plant or specific plant organs in single cell orientation.

Several methods have been developed for multi-class plant hormones analysis, but even the most influential methods are expected to improve the selectivity prior to analyzing different plant hormones isomers belonging to the same class with structural similarities. Recent advancements in the analytical methods with micro-separation ability, excellent selectivity and high sensitivity features would facilitate more comprehensive real-time monitoring of plant hormone during growth and development.

Mass spectrometric analysis is highly suitable for plant hormone analysis and has witnessed widespread application in recent years. Further studies would anticipate to develop derivatization methods for ultra-trace compounds analysis, as derivatization methods improve the analytical sensitivity and the spatial–temporal resolution of plant hormone. Furthermore, the establishment of a mass spectral database for plant hormone is crucial for accurate qualitative and quantitative analysis. Lastly, such a database may act as a tool for advancing scientific theories and for the potential discovery of new plant hormones.

Availability of data and materials

Not applicable.

Abbreviations

Abscisic acid

Gibberellin

Brassinosteroid

Salicylic acid

Jasmonic acid

Strigolactone

Fresh weight

• Mass spectrometry

Gas chromatography–mass spectrometry

Liquid chromatography-mass spectrometry

Quadrupole time-of-flight

Triple quadrupole instruments

Liquid–liquid extraction

Liquid phase microextraction

Dispersive liquid–liquid microextraction

Polymer monolith microextraction

Solid phase extraction

Solid phase microextraction

Magnetic solid phase extraction

Ion pair stir bar sorptive extraction

Electromembrane extraction

Capillary electrophoresis

Dispersive solid-phase extraction

High performance liquid chromatography

Ultra high performance liquid chromatography-mass spectrometry

Reversed phase liquid chromatography

Limit of detection

Limit of quantitation

On-cyclodextrin

Naphthaleneacetic acid

2-Naphthoxyacetic acid

Trans-zeatin

28-Epihomobrassinolide

Poly(methacrylic acid-co-ethylene dimethacrylate)

Relative standard deviation

Electrospray ionization

Atmospheric pressure chemical ionization

Acetonitrile

Triple quadrupole

Time of flight

Quadrupole time of flight

Tandem mass spectrometry

Selected ion monitor mode

Multiple reaction monitor mode

Selected reaction monitor mode

Electrospray ionization mass spectrometry

Quadrupole time-of-flight mass spectrometer

Liquid chromatography-triple quadrupole mass spectrometry

Carboxyl groups

Hydroxyl groups

N -(acridin-9-yl)-2-bromoacetamide

Bromocholinebromide

3-Bromoactonyltrimethylammonium bromide

N -(3-dimethylaminopropyl)- N -ethylcarbodiimide hydrochloride

Solid phase boronate affinity labeling

Salt-induced phase transition extraction

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Acknowledgements

This study was supported by a grant from the National Natural Science Foundation of China (Nos. 21775134, 51703192).

This work was supported by the Open Funds of the State Key Laboratory of Rare Earth Resource Utilization (RERU2017003).

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Liyuan Wang, Yilin Zou, Han Yeong Kaw, Gang Wang, Huaze Sun, Long Cai, Long-Yue Meng & Donghao Li

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Wang, L., Zou, Y., Kaw, H.Y. et al. Recent developments and emerging trends of mass spectrometric methods in plant hormone analysis: a review. Plant Methods 16 , 54 (2020). https://doi.org/10.1186/s13007-020-00595-4

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Analytical Approaches Based on Gas Chromatography Mass Spectrometry (GC/MS) to Study Organic Materials in Artworks and Archaeological Objects

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• Volume 374 , article number  6 , ( 2016 )

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• Ilaria Bonaduce   ORCID: orcid.org/0000-0003-0346-8373 1 ,
• Erika Ribechini 1 ,
• Francesca Modugno 1 &
• Maria Perla Colombini 1 , 2  

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Gas chromatography/mass spectrometry (GC/MS), after appropriate wet chemical sample pre-treatments or pyrolysis, is one of the most commonly adopted analytical techniques in the study of organic materials from cultural heritage objects. Organic materials in archaeological contexts, in classical art objects, or in modern and contemporary works of art may be the same or belong to the same classes, but can also vary considerably, often presenting different ageing pathways and chemical environments. This paper provides an overview of the literature published in the last 10 years on the research based on the use of GC/MS for the analysis of organic materials in artworks and archaeological objects. The latest progresses in advancing analytical approaches, characterising materials and understanding their degradation, and developing methods for monitoring their stability are discussed. Case studies from the literature are presented to examine how the choice of the working conditions and the analytical approaches is driven by the analytical and technical question to be answered, as well as the nature of the object from which the samples are collected.

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Bonaduce, I., Ribechini, E., Modugno, F. et al. Analytical Approaches Based on Gas Chromatography Mass Spectrometry (GC/MS) to Study Organic Materials in Artworks and Archaeological Objects. Top Curr Chem (Z) 374 , 6 (2016). https://doi.org/10.1007/s41061-015-0007-x

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DOI : https://doi.org/10.1007/s41061-015-0007-x

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Gas Chromatography-Mass Spectrometry (GC-MS) Applications

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Gas chromatography–mass spectrometry (GC-MS) is a hybrid analytical technique that couples the separation capabilities of GC with the detection properties of MS to provide a higher efficiency of sample analyses. While GC can separate volatile components in a sample, MS helps fragment the components and identify them on the basis of their mass.

GC-MS provides enhanced sample identification, higher sensitivity, an increased range of analyzable samples, and faster results, which enable a whole new range of applications for GC-MS in several areas.

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Environmental Monitoring

Monitoring environmental pollutants is a major application of GC-MS. It is widely used in the detection of dibenzofurans, dioxins, herbicides, sulfur, pesticides, phenols, and chlorophenols in air, soil, and water.

Food and Fragrance Analysis

Aromatic compounds such as fatty acids, esters, aldehydes, alcohols, and terpenes present in food and beverages can be easily analyzed using GC-MS.  The technique can also be used to detect c spoilage or contamination of food. The analysis of a wide range of oils such as lavender oil, olive oil, spearmint oil, and essential oils, perfumes, fragrances, allergens, menthol, and syrups is also possible using GC-MS.

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In the pharmaceutical industry, GC-MS is used in research and development, production, and quality control. It is used in identification of impurities in active pharmaceutical ingredients. In medicinal chemistry, GC-MS is used in the synthesis and characterization of compounds and in pharmaceutical biotechnology.

Forensic Applications

Using GC-MS, fire debris analysis can be performed as per the American Society for Testing Materials (ASTM) standards. GC-MS is widely used in forensic toxicology to identify poisons and steroids in biological specimens and in anti-doping labs to detect performance enhancing drugs such as anabolic steroids.

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Life science instruments manufacturer, Agilent offers single and triple quadrupole GC-MS  systems that are very sensitive with low detection limits and are suitable for forensics applications, food testing and toxicology analysis.

Biological Analysis

GC-MS can be used for the bioanalysis of body fluids to detect narcotics, barbiturates, alcohols, and drugs such as anticonvulsants, anesthetics, antihistamines, sedative hypnotics, and anti-epileptic drugs. It is also useful in detecting pollutants and metabolites in serum and in fatty acid profiling in microbes.

GC-MS systems from scientific solution provider, Thermo Fischer Scientific are coupled with software that helps streamline GC-MS workflows and data and can be seamlessly integrated with food, environmental, forensic and clinical applications.

Chemical Warfare

Explosive detection systems in public places use GC-MS technique for the analysis and detection of chemical warfare agents.

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Due to its structurally significant mass spectral peaks, extended range of analyzable low volatility samples, enhanced molecular ions, and valuable isotope ratio information, GC-MS is a powerful tool for geochemical applications. GC-MS has been used to analyze the atmosphere of Venus and has also been used by the Viking program on Mars. Additionally, a chiral GC-MS system has been used by the Rosetta mission to analyze the materials in the comet 67P/Churyumov-Gerasimenko.

Industrial Applications

GC-MS is ideal for the analysis of inorganic gases and aromatic solvents, detection of impurities and allergens in cosmetics.  It is also used in the synthesis of cellulose acetate, polyethylene, polyvinyl, and synthetic fibers.

Therefore we may conclude that automated GC-MS systems offer rapid and reproducible results in several applications.

• http://www.ijpba.info/ijpba/index.php/ijpba/article/download/464/314
• http://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.php?aid=33334
• https://www.thermofisher.com/ca/en/home/industrial/mass-spectrometry/gas-chromatography-mass-spectrometry-gc-ms.html
• https://www.agilent.com/
• http://www.perkinelmer.com/category/gas-chromatography-mass-spectrometry-gc-ms

Further Reading

• All Chromatography Content
• Chromatography Overview
• High Performance Liquid Chromatography (HPLC)
• Liquid Chromatography-Mass Spectrometry (LC-MS) Applications
• Affinity Chromatography – How Does it Work?

Last Updated: Jul 24, 2023

Susha Cheriyedath

Susha is a scientific communication professional holding a Master's degree in Biochemistry, with expertise in Microbiology, Physiology, Biotechnology, and Nutrition. After a two-year tenure as a lecturer from 2000 to 2002, where she mentored undergraduates studying Biochemistry, she transitioned into editorial roles within scientific publishing. She has accumulated nearly two decades of experience in medical communication, assuming diverse roles in research, writing, editing, and editorial management.

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