Journal of Chromatography B (v.992, #C)
Editorial Board (i).
Simultaneous determination of four designer drugs and their major metabolites by liquid chromatography–mass spectrometry by Xueguo Chen (1-7).
A sensitive liquid chromatography–electrospray ionization-ion trap mass spectrometry (LC–ESI-ITMS) method was utilized for the simultaneous analysis of four designer drugs and their in vitro metabolites in rat liver microsome S9 fraction. Four designer drugs, including methcathinone (MC), 3,4-methylenedioxymethcathinone (MDMC), 3,4-methylenedioxy-pyrovalerone (MDPV) and 4′-methyl-α-pyrrolidinopropiophenone (MPPP), were individually incubated with rat liver microsome S9 fraction, and the incubation mixtures were pooled together and analyzed by LC–ESI-ITMS simultaneously. Besides four designer drugs, five of their main metabolites were identified via the analysis of protonated molecules and tandem mass spectrometry data. Meanwhile, the quantification analysis of four designer drugs in rat liver microsome S9 fraction was performed, the calibration curves showed good linearity in the range of 0.01–5.0 μg/mL and the detection limits were below 0.03 μg/mL with RSDs less than 5.9% and recovery ratios above 77.4%. The experimental results not only showed that these designer drugs could be easily metabolized in rat liver microsome, and also displayed the superiorities of the method including time and cost saving, high efficiency, sensitivity and selectivity. The studies in this study indicated that the approach could be applied in the determination of illicit drugs and their metabolites in medical, pharmaceutical and forensic investigations.
Keywords: Liquid chromatography–mass spectrometry; Rat liver microsome; Designer drug; Metabolism;
Simultaneous determination of plant hormones in peach based on dispersive liquid–liquid microextraction coupled with liquid chromatography–ion trap mass spectrometry by Qiaomei Lu; Wenmin Zhang; Jia Gao; Minghua Lu; Lan Zhang; Jianrong Li (8-13).
Fruit development is influenced greatly by endogenous hormones including salicylic acid (SA) and abscisic acid (ABA). Mass spectrometry with high sensitivity has become a routine technology to analyze hormones. However, pretreatment of plant samples remains a difficult problem. Thus, dispersive liquid–liquid microextraction (DLLME) was used to concentrate trace plant hormones before liquid chromatography–ion trap mass spectrometry (LC–ITMS) analysis. Standard curves were linear within the ranges of 0.5–50, 0.2–20 ng/mL for SA and ABA, respectively. The correlation coefficients were greater than 0.9995 with recoveries above 87.5%. The limits of detection were 0.2 ng/mL for SA and 0.1 ng/mL for ABA in spiked water solution, respectively (injection 20 μL). The successful analysis of SA and ABA in fruit samples indicated our DLLME–LC–ITMS approach was efficient, allowing reliable quantification of both two compounds from very small amounts of plant material. Moreover, this research revealed the relationship between SA and ABA content and development of peach fruit at different growth stages.
Keywords: Dispersive liquid–liquid microextraction; High performance liquid chromatography; Mass spectrometry; Salicylic acid; Abscisic acid; Plant hormone;
Rapid determination of X-ray contrast agent iomeprol in human plasma by on-line solid-phase extraction coupled with phase optimized liquid chromatography by Tingting Zhou; Yun Tao; Yabing Pu; Rosa Morello; Surong Mei; Karl-Siegfried Boos (14-22).
Phase optimized liquid chromatography (POPLC) allows for the optimized combination of column segments of any length and stationary phases with different functionalities. In this study, a simple and rapid method using POPLC coupled with on-line solid-phase extraction (SPE) for the analysis of X-ray contrast media agent iomeprol (IOM) in human plasma was developed. Because the phenyl (PH) stationary phase has strong hydrophobic and π–π interactions with IOM and iopromide (IOP, internal standard), the best separation efficiency was achieved with a 250 mm × 3 mm homogenous PH POPLC-column. Different kinds of on-line SPE sorbents were studied, including restricted access material-alkyl diol silica (ADS), LiChrolut EN with excellent absorption capacity and hydrophilic–lipophilic-balanced Oasis HLB. The most efficient on-line sample clean-up was carried out using a fast-flow on-line purification approach with an Oasis HLB pre-column ((20 mm × 2 mm, 30 μm). This pre-column showed excellent durability and reproducibility. At least 400 samples could be analyzed with one pre-column. Each plasma sample was directly injected and analyzed within 15 min. The calibration curves were linear in the range of 10–1000 μg/mL. The limit of quantitation was 2.26 μg/mL. The inter-day precision of this method was excellent and less than 1.44%, and the intra-day precision was less than 4.44%. The inter-day and intra-day accuracy ranged from 94.33% to 104.36% and 94.60% to 101.71%, respectively. This validated method is expected to be useful in the analysis of human plasma samples for glomerular filtration rate (GFR) measurements and assessment of kidney function.
Keywords: Iomeprol; Phase optimized liquid chromatography; On-line SPE; Plasma;
One-step purification of R-phycoerythrin from the red edible seaweed Grateloupia turuturu by Mathilde Munier; Michèle Morançais; Justine Dumay; Pascal Jaouen; Joël Fleurence (23-29).
A one-step chromatographic method for the purification of R-phycoerythrin (R-PE) of Grateloupia turuturu Yamada is described. Native R-PE was obtained with a purity index of 2.89 and a recovery yield of 27% using DEAE-Sepharose Fast Flow chromatography with a three-step increase in ionic strength. The analysis by SDS electrophoresis showed a broad band between 18 and 21 kDa in size corresponding to subunits α and β and a low intensity band of 29 kDa corresponding to the γ subunit. Two forms of R-PE were identified by gel filtration chromatography: a native form with a molecular weight of 260 ± 5 kDa and a dissociated form with a molecular weight of 60 ± 2 kDa. The native form presented the characteristic absorption spectrum of R-PE with three absorbance maxima at 498, 540 and 565 nm, whereas the dissociated form presented only the 498 and 540 nm peaks. Moreover, the two forms displayed two different fluorescence maxima.
Keywords: Grateloupia turuturu; R-phycoerythrin; Purification; Ion-exchange chromatography;
Simultaneous quantification of preactivated ifosfamide derivatives and of 4-hydroxyifosfamide by high performance liquid chromatography–tandem mass spectrometry in mouse plasma and its application to a pharmacokinetic study by Alain Deroussent; Charles Skarbek; Adeline Maury; Hubert Chapuis; Estelle Daudigeos-Dubus; Ludivine Le Dret; Sylvère Durand; Patrick Couvreur; Didier Desmaële; Angelo Paci (30-35).
The antitumor drug, ifosfamide (IFO), requires activation by cytochrome P450 (CYP) to form the active metabolite, 4-hydroxyisfosfamide (4-OHIFO), leading to toxic by-products at high dose. In order to overcome these drawbacks, preactivated ifosfamide derivatives (RXIFO) were designed to release 4-OHIFO without CYP involvement. A high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the simultaneous quantification of 4-OHIFO, IFO and four derivatives RXIFO in mouse plasma using multiple reaction monitoring. Because of its instability in plasma, 4-OHIFO was immediately converted to the semi-carbazone derivative, 4-OHIFO-SCZ. For the six analytes, the calibration curves were linear from 20 to 5000 ng/mL in 50 μL plasma and the lower limit of quantitation was determined at 20 ng/mL with accuracies within ±10% of nominal and precisions less than 12%. Their recoveries ranged from 62 to 96% by using liquid–liquid extraction. With an improved assay sensitivity compared to analogues, the derivative 4-OHIFO-SCZ was stable in plasma at 4 °C for 24 h and at −20 °C for three months. For all compounds, the assay was validated with accuracies within ±13% and precisions less than 15%. This method was applied to a comparative pharmacokinetic study of 4-OHIFO from IFO and three derivatives RXIFO in mice. This active metabolite was produced by some of the novel conjugates with good pharmacokinetic properties.
Keywords: Preactivated ifosfamide; 4-Hydroxyifosfamide; HPLC–MS/MS; Mouse plasma; Pharmacokinetics;
A general separation method of phenolic acids using pH-zone-refining counter-current chromatography and its application to oat bran by Genlai Dong; Jiangning Xu; Yanxiang Gu; Yun Wei (36-42).
pH-zone-refining counter-current chromatography technique for the separation of natural and synthetic mixtures has been widely used, especially for organic acids and alkaloids. Phenolic acids are very important compounds due to the potential treatment for a wide variety of diseases. However, there is not a general method for their separation. In this work, the conditions of pH-zone-refining counter-current chromatography, involving solvent systems, concentration of retainer and eluter, flow rate of mobile phase as well as sample pretreatment, were optimized to improve extraction efficiency and reduce separation time. Finally a general separation method for seven common phenolic acids has been established using pH-zone-refining counter-current chromatography. The separation of these phenolic acids was performed with a two-phase solvent system composed of methyl tert-butyl ether/acetonitrile/water at a volume ratio of 4.75: 0.25: 5, where 3 mM trifluoroacetic acid was added to the organic stationary phase as a retainer and 3 mM NH4OH was added to the aqueous mobile phase as an eluter. As a result, seven phenolic acids, including syringic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, p-hydroxybenzoic acid, ferulic acid and p-coumaric acid were successfully separated with the purities of 95.9%, 67.3%, 96.9%, 82.4%, 97.0%, 91.0%, and 97.2%, respectively. The established general method has been applied to the crude sample of oat bran pretreated with AB-8 resin. A total of 49.5 mg of syringic acid, 109.2 mg of p-coumaric acid and 184.5 mg of ferulic acid were successfully purified in one run from 1.22 g crude extract with the purities of 95.2%, 93.0%, and 91.8%, respectively.
Keywords: Phenolic acids; Oat bran; pH-zone-refining counter-current chromatography;
Quantification of liensinine in rat plasma using ultra-performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study by Song-feng Lv; Xiang-hong Wang; Hong-wei Li; Xiao-lei Zhang; Bo Wang (43-46).
An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine liensinine in rat plasma using carbamazepine as the internal standard (IS). Sample preparation was accomplished through a protein precipitation procedure with acetonitrile to 0.1 ml plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 0.1% formic acid in water with gradient elution at a flow rate of 0.40 ml/min. The injection volume was 6 μl. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 611.6 → 206.2 for liensinine and m/z 237.1 → 194.2 for IS. The linearity of this method was found to be within the concentration range of 10–1000 ng/ml with a lower limit of quantification of 10 ng/ml. Only 3.0 min was needed for an analytical run. The matrix effect was 93.8–107.4% for liensinine. The intra- and inter-day precision (RSD %) were less than 9.9% and accuracy (RE %) was within ±10.5%. The recovery ranged from 76.2 to 86.8%. Liensinine was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of liensinine in rats. The pharmacokinetic parameters were demonstrated as followed: t 1/2 was 8.2 ± 3.3 h, C max was 668.4 ± 156.9 ng/ml, and AUC0→∞ was 1802.9 ± 466.4 ng/ml h.
Keywords: Liensinine; UPLC–MS/MS; Rat plasma; Pharmacokinetics;
New method for the determination of parabens and bisphenol A in human milk samples using ultrasound-assisted extraction and clean-up with dispersive sorbents prior to UHPLC–MS/MS analysis by R. Rodríguez-Gómez; N. Dorival-García; A. Zafra-Gómez; F.J. Camino-Sánchez; O. Ballesteros; A. Navalón (47-55).
A sensitive and accurate analytical method for the determination of methyl-, ethyl-, propyl- and butylparaben and bisphenol A in human milk samples has been developed and validated. The combination of ultrasound-assisted extraction (UAE) and a simplified and rapid clean-up technique that uses sorbent materials has been successfully applied for the preparation of samples prior to ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis. The analytes were extracted from freeze–dried human milk samples using acetonitrile and ultrasonic radiation (three 15-min cycles at 70% amplitude), and further cleaned-up with C18 sorbents. The most influential parameters affecting the UAE method and the clean-up steps were optimized using design of experiments. Negative electrospray ionization (ESI) in the selected reaction monitoring (SRM) mode was used for MS detection. The use of two reactions for each compound allowed simultaneous quantification and identification in one run. The analytes were separated in less than 10 min. Deuterium-labeled ethylparaben-d5 (EPB-d5) and deuterium-labeled bisphenol A-d16 (BPA-d16) were used as surrogates. The limits of quantification ranged from 0.4 to 0.7 ng mL−1, while inter- and intra-day variability was under 11.1% in all cases. In the absence of certified reference materials, recovery assays with spiked samples using matrix-matched calibration were used to validate the method. Recovery rates ranged from 93.8% to 112.2%. The proposed method was satisfactorily applied for the determination of four selected parabens and bisphenol A in human milk samples obtained from nursing mothers living in the province of Granada (Spain).
Keywords: Human milk; Endocrine disrupting chemicals; Ultrasound-assisted extraction; Sorbent materials; UHPLC–MS/MS;
Effective application of freezing lipid precipitation and SCX-SPE for determination of pyrrolizidine alkaloids in high lipid foodstuffs by LC-ESI-MS/MS by Soo Hwan Yoon; Min-Sun Kim; Sang Hoon Kim; Hyun Mee Park; Heesoo Pyo; Yong Moon Lee; Kyung-Tae Lee; Jongki Hong (56-66).
Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with serious hepatic disease in humans and animals. In this study, rapid and sensitive analytical method was developed for the determination of 9 toxic PAs in popularly high lipid foodstuffs by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). PAs in lipid foodstuff were effectively purified by freezing lipid precipitation (FLP) and strong cation exchange (SCX)-solid-phase extraction (SPE). Especially, FLP could easily remove the large amounts of triacylglycerols in the lipid sample extract and effectively combine with SPE cleanup. During the FLP procedure, over 77% of the lipids in the foodstuff extracts were rapidly eliminated without any significant loss of the PAs with over 81% recovery. The elimination efficiency of lipids by FLP was tested with LC-atmospheric chemical ionization (APCI)-MS. For further purification, SCX-SPE cartridge could successfully purify PAs from the remaining interfering substances by the variation pH with 5% NH4OH in methanol. For precise quantification and confirmation of PAs in complicate sample matrices, appropriate transition ions in LC-MS/MS-multiple-ion reaction monitoring (MRM) mode were selected on the basis of MS/MS fragmentation pathways of PAs. The established analytical method was validated in terms of the linearity, limits of detection (LOD), and quantification (LOQ), precision, and accuracy. The method was deemed satisfactory by inter- and intra-day validation and exhibited both high accuracy and precision (relative standard deviation < 11.06%). Overall limits of detection and quantitation of PAs were approximately 0.06–0.60 ng/mL at a signal-to-noise ratio (S/N) of 3 and were about 0.20–1.99 ng/mL at a S/N of 10 for all foodstuffs. The established method was successfully applied for the monitoring of toxic PAs in several types of high lipid foodstuffs such as soybeans, seed oil, milk, and margarine.
Keywords: Pyrrolizidine alkaloids; High lipid foodstuffs; Freezing lipid precipitation; SCX-SPE; LC-ESI-MS/MS-MRM;
Critical development by design of a rugged HPLC-MS/MS method for direct determination of ibuprofen enantiomers in human plasma by Natalija Nakov; Rumenka Petkovska; Liljana Ugrinova; Zoran Kavrakovski; Aneta Dimitrovska; Dobrin Svinarov (67-75).
Development and validation of a HPLC-MS/MS method for direct determination of R- and S-ibuprofen (Ibu) in human plasma without a need of derivatization or other complexities such as postcolumn infusion of solvents or reagents was performed. Critical steps were investigated during method development using experimental design to achieve a reliable and rugged assay. The LC-MS/MS separation of R-Ibu and S-Ibu was obtained on Lux Cellulose chiral column utilizing 0.1% (v/v) acetic acid in mixture of methanol and water (90:10%, v/v) as a mobile phase. Two types of extraction procedure for Ibu and Ketoprofen (internal standard, IS) were optimized using Full factorial 32 design (LLE) and D-Optimal Experimental Design (SPE). Excellent recovery values, 80% (mean) and 95% (mean) for LLE and SPE respectively, were obtained using 50 μL plasma. The matrix effect was assessed for both of the extraction procedures, including hyperlipidaemic and haemolyzed plasma. The extensive investigation of matrix effect showed that LLE yields cleaner extracts than the SPE. The result of the investigation of in vitro interconversion of R-Ibu and S-Ibu showed that it does not occur under the influence of pH, temperature, and in the overall analytical procedure. The validation data, adhered to EMA guideline for validation of bioanalytical methods, showed that the proposed method provides accurate and reproducible results in range of 0.1–50 mg/L with a lower limit of detection of 0.02 mg/L. The applicability of the method was demonstrated through determination of R-Ibu and S-Ibu in human plasma after oral administration of 400 mg rac-Ibu.
Keywords: Ibuprofen enantiomers; LC-MS/MS; Sample preparation; Experimental design; Matrix effect; in vitro Chiral interconversion;
A method for quantification of volatile organic compounds in blood by SPME-GC–MS/MS with broader application: From non-occupational exposure population to exposure studies by Rocio Aranda-Rodriguez; Ashley Cabecinha; Jeromy Harvie; Zhiyun Jin; Axelle Marchand; Robert Tardif; Andy Nong; Sami Haddad (76-85).
Humans are continuously exposed to volatile organic compounds (VOCs) as these chemicals are ubiquitously present in most indoor and outdoor environments. In order to assess recent exposure to VOCs for population-based studies, VOCs are measured in the blood of participants. This work describes an improved method to detect 12 VOCs by head-space solid-phase microextraction gas chromatography coupled with isotope-dilution mass spectrometry in selected reaction monitoring mode (SPME-GC–MS/MS). This method was applied to the analysis of trihalomethanes, styrene, trichloroethylene, tetrachloroethylene and BTEX (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene) in a population-based biomonitoring study (Canadian Health Measures Survey). The method showed good linearity (>0.990) in the range of 0.010–10 μg/L and detection limits between 0.007 and 0.027 μg/L, precision better than 25% and good accuracy (±25%) based on proficiency testing materials. Quality Control data among runs over a 7 month period showed %RSD between 14 and 25% at low levels (∼0.03 μg/L) and between 9 and 23% at high levels (∼0.4 μg/L). The method was modified to analyze samples from a pharmacokinetic study in which 5 healthy volunteers were exposed to single, binary and quaternary mixtures of CTEX (chloroform, ethylbenzene, toluene and m-xylene), thus the expected concentration in blood was 1 order of magnitude higher than those found in the general population. The method was modified by reducing the sample size (from 3 g to 0.5 g) and increasing the upper limit of the concentration range to 395 μg/L. Good linearity was found in the range of 0.13–395 μg/L for toluene and ethylbenzene and 0.20–609 μg/L for m/p-xylene. Quality control data among runs over the period of the study (n = 13) were found to vary between 7 and 25%.
Keywords: Biomonitoring; Blood; Volatile organic compounds; Solid-phase microextraction; General population; Exposure studies;
Graphite-based microextraction by packed sorbent for online extraction of β-blockers from human plasma samples by Thana Abuzooda; Ahmad Amini; Mohamed Abdel-Rehim (86-90).
In the present work a new graphitic material (Carbon-XCOS) was used as a sorbent for microextraction by packed sorbent (MEPS). The β-blockers metoprolol and acebutolol in plasma samples were extracted and detected online using Carbon-MEPS syringe and liquid chromatography and tandem mass spectrometry (LC–MS/MS). Factors affecting the MEPS performance such as conditioning, washing and elution solutions were investigated. The validation of the bioanalytical method was performed using human plasma. The standard curve ranged from 10 to 2000 nM and the lower limit of quantification (LLOQ) was set to 10 nM. The method validation showed good accuracy and precision for the quality control (QC) samples at three concentration levels (30, 800 and 1600 nM). The accuracy values of the QC samples were in the range of 86–108% (n = 18). The precision values of intra- and inter-day for QC samples ranged from 4.4% to 14.4% (RSD) for the both studied analytes. The coefficient of determination (R 2) values were ≥0.999 (n = 3).
Keywords: Graphitic sorbent; Microextraction by packed sorbent; β-Blocker; Plasma samples;
Simultaneous determination of acetaminophen and dihydrocodeine in human plasma by UPLC–MS/MS: Its pharmacokinetic application by Xiangjun Qiu; Dan Lou; Ding Su; Zebin Liu; Pengtao Gao; Nan-sheng Zhang (91-95).
An ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to determine acetaminophen (AAP) and dihydrocodeine (DHC) in human plasma simultaneously. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard midazolam were separated on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 151.2 → 110.0 and m/z 302.3 → 199.2 were used to quantify for AAP and DHC, respectively. The linearity of this method was found to be within the concentration range of 50–10000 ng/mL for AAP, and 1–100 ng/mL for DHC in human plasma, respectively. The lower limit of quantification (LLOQ) was 50 ng/mL and 1 ng/mL for AAP and DHC in human plasma, respectively. The relative standard deviations (RSD) of intra and inter precision were less than 10% for both AAP and DHC. The analysis time of per sample was 1.0 min. The developed and validated method was successfully applied to a pharmacokinetic study of AAP (500 mg) with DHC (20 mg) capsule in Chinese healthy volunteers (N = 20).
Keywords: Acetaminophen; Dihydrocodeine; UPLC–MS/MS; Human plasma; Pharmacokinetic;
Simultaneous determination of polycyclic musks in blood and urine by solid supported liquid–liquid extraction and gas chromatography–tandem mass spectrometry by Hongtao Liu; Liping Huang; Yuxin Chen; Liman Guo; Limin Li; Haiyun Zhou; Tiangang Luan (96-102).
A rapid, precise and accurate method for the simultaneous determination of 5 polycyclic musks (PCMs) in biological fluids was developed by solid supported liquid–liquid extraction (SLE) coupled with gas chromatography–tandem mass spectrometry (GC–MS/MS). All parameters influencing SLE-GC–MS performance, including electron energy of electron-impact ionization source, collision energy for tandem mass spectrometer when operated in selected-reaction monitoring (SRM) mode, type and volume of elution reagent, nitrogen evaporation time, pH and salinity of sample have been carefully optimized. Eight milliliter of n-hexane was finally chosen as elution reagent. Blood and urine sample could be loaded into SLE cartridge without adjusting pH and salinity. Deuterated tonalide (AHTN-d3) was chosen as internal standard. The correlation coefficient (r 2 ) of the calibration curves of target compounds ranged from 0.9996 to 0.9998. The dynamic range spanned over two orders of magnitude. The limit of detection (LOD) of target compounds in blood and urine ranged from 0.008 to 0.105 μg L−1 and 0.005 to 0.075 μg L−1, respectively. The developed procedure was successfully applied to the analysis of PCMs in human blood and urine obtaining satisfying recoveries on low, medium and high levels. The method was compared with SLE-GC–MS and shown one to two orders of magnitude improvement in sensitivity.
Keywords: Polycyclic musks (PCMs); Pharmaceuticals and personal care products (PPCPs); Solid supported liquid–liquid extraction (SLE); Gas chromatography–tandem mass spectrometry (GC–MS/MS);
Determination of 3-mercaptopropionic acid by HPLC: A sensitive method for environmental applications by P. Salgado; T. Visnevschi-Necrasov; R.P. Kiene; I. Azevedo; A.C.S. Rocha; C.M.R. Almeida; C. Magalhães (103-108).
The organic sulfur compound 3-mercaptopropionic acid (3-MPA) is an important thiol intermediate in organic sulfur metabolism in natural environments. It is generated during degradation of sulfur-containing amino acids (e.g. methionine) and from demethylation of dimethylsulfoniopropionate (DMSP). This pathway is an alternative enzymatic process in the DMSP catabolism that routes sulfur away from the climatically-active dimethyl sulfide (DMS). 3-MPA detection and subsequent quantification in different matrices is difficult due to its extreme reactivity. We therefore developed a sensitive method for determination of 3-MPA based on pre-column derivatization with monobromobimane and analysis by high-performance liquid chromatography (HPLC) with fluorescence detection. This methodology was first tested with 3-MPA standards under low (0.005–0.2 μmol L−1) and high (1–25 μmol L−1) concentrations. For the optimization of the reaction, CHES and, alternatively, Tris–HCl buffers were evaluated in the derivatization step, with Tris–HCl showing more effective separation of thiol derivatives and a better 3-MPA peak shape. The detection limit was 4.3 nmol L−1 with a 10 μL sample injection, and mean recoveries of 3-MPA ranged from 97 to 105% in estuarine waters with different salinities (0.17 and 35.9 ppt). The linearity (r > 0.99) and repeatability of detector response, with intra- and inter-day precision (% CV) of 2.68–7.01% and 4.86–12.5%, respectively, confirmed the reliability of the method. Previous 3-MPA analytical methods required immediate analysis due to unstable derivatives, but in this method we achieved high stability of the derivatized samples when stored at 4 °C, with only a 3–5% loss after more than one year of storage. This method was successfully applied to measure 3-MPA concentrations and rates of 3-MPA production in a variety of intertidal estuarine sediment slurries. Dissolved 3-MPA concentrations in these sediment slurries varied between 2 and 237 μmol L−1 and, 3-MPA net fluxes ranged in wet sediments between −3.6 ± 1.7 and 30 ± 5 μmol L−1 g−1 h−1. Thus, the application of this optimized methodology showed an efficient performance for measuring 3-MPA in environmental samples, with a straightforward sample derivatization and a simple analysis of stable 3-MPA derivatives.
Keywords: 3-Mercaptopropionic acid (3-MPA); High-Performance Liquid Chromatography (HPLC); Monobromobimane; Thiols;