Development of a PDMS‐based microchip electrophoresis device for continuous online in vivo monitoring of microdialysis samples

A PDMS‐based microfluidic system for online coupling of microdialysis sampling to microchip electrophoresis with fluorescence detection for in vivo analysis of amino acid neurotransmitters using naphthalene‐2,3‐dicarboxaldehyde and sodium cyanide as the derivatization reagents is described. Fabricating chips from PDMS rather than glass was found to be simpler and more reproducible, especially for chips with complex designs. The microchip incorporated a 20‐cm serpentine channel in which sample plugs were introduced using a “simple” injection scheme; this made fluid handling and injection on‐chip easier for the online system compared with gated or valve‐based injection. The microchip was evaluated offline for the analysis of amino acid standards and rat brain microdialysis samples. Next, precolumn derivatization was incorporated into the chip and in vivo online microdialysis‐microchip electrophoresis studies were performed. The system was employed for the continuous monitoring of amino acid neurotransmitters in the extracellular fluid of the brain of an anesthetized rat. Fluorescein was dosed intravenously and monitored simultaneously online as a marker of in vivo blood–brain barrier permeability. The microdialysis‐microchip electrophoresis system described here will be employed in the future for simultaneous monitoring of changes in blood–brain barrier permeability and levels of amino acid neurotransmitters in the rat stroke model.     

雄黄染毒后大鼠脑组织中氨基酸类神经递质含量的变化

研究了雄黄对大鼠脑组织氨基酸类神经递质含量的影响.将32只Wistar大鼠随机分为对照组(0.5%CMC-Na)以及低剂量(0.3g/kg)、中剂量(0.9g/kg)、高剂量(2.7g/kg)雄黄染毒组,通过连续灌胃给予雄黄混悬液两周.采用高效液相色谱-柱前衍生化法测定了大鼠脑组织中氨基酸类神经递质含量的变化.结果表明,与对照组比较,低剂量组大鼠脑组织中丝氨酸、甘氨酸和γ-氨基丁酸含量明显增加.中、高剂量组大鼠脑组织中同型半胱氨酸、谷氨酰胺、丝氨酸和天冬氨酸含量明显比对照组的低.总体而言,雄黄可对大鼠脑组织氨基酸类神经递质产生影响,氨基酸类神经递质可能是雄黄毒性作用的靶点之一.     

Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o‐phthalaldehyde

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high‐throughput analysis with flow‐gated CE. Here, we report a fast fluorescein‐labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow‐gated CE. This scheme was based on the reaction between primary amines and o‐phthalaldehyde in the presence of a fluorescent thiol, 2‐((5‐fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE‐SH). The short reaction time (<30 s) was suited for on‐line mixing and derivatization that was directly coupled with flow‐gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE‐SH, Tris(2‐carboxyethyl)phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow‐gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on‐chip labeling of proteins retained on support in SPE.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

A derivatization and validation strategy for determining the spatial localization of endogenous amine metabolites in tissues using MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here, we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix‐assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4‐hydroxy‐3‐methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high‐mass resolution and MSⁿ IMS. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high‐performance liquid chromatography (HPLC)‐MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a fluorimetric detection method for cinnabarinic acid using ortho‐tolyl hydrazine as the derivatization reagent

A fluorimetric detection method for one of the tryptophan metabolites, cinnabarinic acid (CA), which has recently been reported to have the ability to induce apoptosis in thymocytes, was developed using o‐tolyl hydrazine (TH) as the derivatization reagent. The carbonyl group at position 3 in CA was tagged with the hydrazino moiety of TH at 100°C for 30 min, and the generated derivative, CA tagged with TH, fluoresced at 412 nm with a 316 nm excitation wavelength. The CA tagged with TH was separated on a reversed‐phase HPLC and detected fluorometrically. The relative standard deviation was in the range of 1.1–8.9% (n = 3), and the detection limit was approximately 12?fmol (signal‐to‐noise ratio, 3). The proposed HPLC method can be useful for the sensitive detection of CA. Copyright © 2009 John Wiley & Son, Ltd.     

Convenient One‐Pot Synthesis of α‐Amino Phosphonates in Water Using p‐Toluenesulfonic Acid as Catalyst for the Kabachnik–Fields Reaction

The three‐component Kabachnik–Fields reaction of substituted salicylaldehydes, aromatic amine, and triphenyl phosphite in water was effectively catalyzed by p‐toluenesulfonic acid to give various α‐amino phosphonates in good yields. The catalyst is easily available and inexpensive, and the process is green and mild. © 2013 Wiley Periodicals, Inc. Heteroatom Chem 24:110–115, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/hc.21071     

Reactivity of Tyr–Leu and Leu–Tyr dipeptides: identification of oxidation products by liquid chromatography–tandem mass spectrometry

The exposure of peptides and proteins to reactive hydroxyl radicals results in covalent modifications of amino acid side‐chains and protein backbone. In this study we have investigated the oxidation the isomeric peptides tyrosine–leucine (YL) and leucine–tyrosine (LY), by the hydroxyl radical formed under Fenton reaction (Fe²⁺/H₂O₂). Through mass spectrometry (MS), high‐performance liquid chromatography (HPLC‐MS) and electrospray tandem mass spectrometry (HPLC‐MSⁿ) measurements, we have identified and characterized the oxidation products of these two dipeptides. This approach allowed observing and identifying a wide variety of oxidation products, including isomeric forms of the oxidized dipeptides. We detected oxidation products with 1, 2, 3 and 4 oxygen atoms for both peptides; however, oxidation products with 5 oxygen atoms were only present in LY. LY dipeptide oxidation leads to more isomers with 1 and 2 oxygen atoms than YL (3 vs 5 and 4 vs 5, respectively). Formation of the peroxy group occurred preferentially in the C‐terminal residue. We have also detected oxidation products with double bonds or keto groups, dimers (YL–YL and LY–LY) and other products as a result of cross‐linking. Both amino acids in the dipeptides were oxidized although the peptides showed different oxidation products. Also, amino acid residues have shown different oxidation products depending on the relative position on the dipeptide. Results suggest that amino acids in the C‐terminal position are more prone to oxidation. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of β‐aminopropionitrile,an inhibitor of lysyl oxidase activity,in plasma and tumor of mice by liquid chromatography tandem mass spectrometry

Lysyl oxidase enzymes are reported to be involved in patho‐physiological process such as tumorigenesis. β‐Aminopropionitrile (BAPN) is an irreversible inhibitor of lysyl oxidase activity, suggesting a potentially useful therapeutic of interest in oncology. This paper describes the first assay concerning the quantification of BAPN by mass spectrometry. A high‐performance liquid chromatography tandem mass spectrometry (LC‐MS/MS) assay was developed for the quantification of BAPN in plasma and tumor of mice. This method combines dansyl chloride (Dns) derivatization and extraction using a solid‐phase extraction Oasis^© Max column. Deuterated BAPN was used as internal standard (IS). Separation was achieved using an C₁₈ column HypersylGold, (ThermoElectron), 3.0 µm (100 × 2.1 mm i.d.). Gradient elution with water containing 0.1% acetic acid (A) and acetonitrile containing 0.1% acetic acid (B) was applied. Detection was performed with an electrospray ionization interface operating in negative ion mode. Selected reaction monitoring was used with ion transitions m/z 302 → 249 for BAPN–Dns and m/z 306 → 250 for the IS. The method was fully validated in plasma and was linear and sensitive in the range of 10–500 ng/mL. The lower limit of quantification in plasma was 2.5 ng/mL. This validated assay was successfully applied to a kinetic study of BAPN in mouse plasma and demonstrates that BAPN reaches the tumoral tissue. Copyright © 2014 John Wiley & Sons, Ltd.     

A proteomics method using immunoaffinity fluorogenic derivatization–liquid chromatography/tandem mass spectrometry (FD‐LC‐MS/MS) to identify a set of interacting proteins

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD‐LC–MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC–tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90‐beta. The results demonstrated that the proposed immunoaffinity–FD‐LC–MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein.     

Optimization and validation of an SBSE–HPLC–FD method using laboratory‐made stir bars for fluoxetine determination in human plasma

Depression is the largest cause of disability worldwide, affecting 350 million people. Notwithstanding that clinical trials demonstrate antidepressants efficacy, the efficient response can vary individually concerning therapeutic dosage. Although important, plasma levels monitoring remains an analytical challenge whereas clean‐up and pre‐concentration represent critical steps. Therefore, this study aims to develop, optimize and validate a method for fluoxetine determination in human plasma, employing a laboratory‐made device consisting of a PDMS stir bar sorptive for extraction, coupled with high‐performance liquid chromatography–fluorescence detection (SBSE–HPLC–FD). Optimization involved sorption–desorption steps. For sorption, temperature and time were assessed by factorial and central composite design approaches, taking into account the desirability and the response surface results, with stirring speed also examined. For desorption kinetics and ultrasonic and magnetic stirring mode were evaluated. The proposed method after validation was robust, linear (25.00–1000.00 ng mL^?1, R² > 0.98) and presented good intra‐ (RSD 4.18%) and inter‐day‐assay (RSD 11.60%) precision and accuracy (recovery 109.60%), allowing reliable quantitation without interference. The method was successfully applied to real samples. SBSE–HPLC–FD could represent a feasible alternative with good cost–benefit for low‐volume samples and therapeutic drug monitoring, as well as contributing to correlation studies between plasma fluoxetine levels and clinical response, which is still little studied.     

A novel preventive mechanism of gentamicin‐induced nephrotoxicity by atorvastatin

Atorvastatin (ATO) inhibits the synthesis of nonsteroidal isoprenoid compounds and possesses a pleiotropic effect. However, the detailed mechanism of ATO in preventing gentamicin (GM)‐induced renal injury remains obscure. Although underlying multifaceted mechanisms involving GM‐induced nephrotoxicity were well known, further work on elucidating the essential mechanism was needed. Using a fluorogenic derivatization–liquid chromatography tandem mass spectrometry proteomic method (FD‐LC–MS/MS method), we investigated the effects and mechanisms of ATO treatment on GM‐induced nephrotoxicity in rats. Consequently, 49 differentially expressed proteins were identified. The most significant mechanisms of nephrotoxicity caused by GM were mitochondrial dysfunction, fatty acid metabolism and oxidative stress. Their upstream regulator was found to be PPARα. The proteins involved in GM nephrotoxicity were sodium–hydrogen exchanger regulatory factor (SLC9A3R1), cathepsin V (CTSV), macrophage migration inhibitory factor (MIF) and RhoGDP dissociation inhibitor alpha (ARHGDIA). After ATO intervention, we observed a reversed enrichment pattern of their expression, especially in CTSV and SLC9A3R1 (P‐value<0.05). We predicted that ATO may improve abnormal phospholipid metabolism and phospholipidosis caused by GM and also alleviate cell volume homeostasis and reverse the interference of GM with the transporter. Furthermore, proteomic results also provided clues as to GM‐induced nephrotoxicity biomarkers such as CTSV and transthyretin.     

Development,validation and comparison of surrogate matrix and surrogate analyte approaches with UHPLC–MS/MS to simultaneously quantify dopamine,serotonin and γ‐aminobutyric acid in four rat brain regions

As biomarkers, endogenous neurotransmitters play critical roles in the process of neuropsychiatric diseases, and neurotransmitter levels in different brain regions can contribute to neurological disease diagnosis and treatment. Due to the lack of a blank matrix for endogenous neurotransmitters, surrogate‐matrix and surrogate‐analyte approaches have been used for the determination of neurotransmitters to solve this problem. In this study, we capitalised on the high accuracy, precision, and throughput of UHPLC‐MS/MS and developed new methods based on the two approaches. Both approaches satisfied FDA and EMA validation criterias after an appropriate parallelism assessment, and they were used to further quantify the three endogenous neurotransmitters, including dopamine (DA), serotonin (5‐HT) and γ‐aminobutyric acid (GABA) in rat brain four regions (cortex, striatum, hypothalamus and hippocampus) which represent the catecholamines, indolamines, and amino acids, respectively. Comparison of the results in the same rats (n = 10) showed there was no significant difference in DA, 5‐HT, or GABA levels between the two approaches (P > 0.05). The concentrations of DA and GABA were highest in striatum and hypothalamus, respectively, and the levels of 5‐HT were paralleled in striatum and hippocampus almost 2‐fold higher than other regions. This is the first study to compare these two approaches in the determination of endogenous neurotransmitter content in the rat brain, and the surrogate‐matrix approach proved to be simple, rapid, and reliable, considering cost, matrix similarity, and practicality.     

The comparative pharmacokinetics of four bioactive ingredients after administration of Ramulus Cinnamomi–Radix Glycyrrhizae herb pair extract,Ramulus Cinnamomi extract and Radix Glycyrrhizae extract

Ramulus Cinnamomi (RC)–Radix Glycyrrhizae (RG) is a classic herb pair, which is commonly used as a fixed form to treat cardiovascular disease in the clinic. Our work aimed to compare the pharmacokinetic difference of cinnamic acid, liquiritin, isoliquiritigenin and glycyrrhetinic acid in rats after oral administration of the RC–RG herb pair extracts [Guizhigancao Decoction (GGD) and Lingguizhugan Decoction (LGZGD)] and the single RC or RG extract. A HPLC‐MS method was developed and validated to study comparative pharmacokinetics. The pharmacokinetic parameters (C_max, AUC, MRT) of four compounds between the RC–RG herb pair group and the single herb (RC or RG) group showed significant differences (p < 0.05). Compared with the single herb (RC or RG) group, higher peak concentration, slower elimination and larger exposure could be observed after giving the RC–RG herb‐pair extracts. The pharmacokinetic differences might indicate the relativity of remedy in the RC–RG herb pair and provide scientific information for rational administration of the drug in the clinic. Copyright © 2016 John Wiley & Sons, Ltd.     

A simple and sensitive high‐performance liquid chromatography–electrochemical detection assay for the quantitative determination of monoamines and respective metabolites in six discrete brain regions of mice

A rapid, sensitive, and reproducible assay is described for the quantitative determination of the monoamine neurotransmitters dopamine, norepinephrine and serotonin, their metabolites, and the internal standard 3,4‐dihydroxybenzlyamine hydro‐bromide in mouse brain homogenate using high‐performance liquid chromatography with electrochemical detection. The method was validated in the following brain areas: frontal cortex, striatum, nucleus accumbens, hippocampus, substantia nigra pars compacta and ventral tegmental area. Biogenic amines and relevant metabolites were extracted from discrete brain regions using a simple protein precipitation procedure, and the chromatography was achieved using a C₁₈ column. The method was accurate over the linear range of 0.300–30 ng/mL (r = 0.999) for dopamine and 0.300–15 ng/mL (r = 0.999) for norepinephrine, 3,4‐dihydroxybenzlyamine hydro‐bromide, homovanillic acid and 5‐hydroxyindolacetic acid, with detection limits of ~0.125 ng/mL (5 pg on column) for each of these analytes. Accuracy and linearity for serotonin were observed throughout the concentration range of 0.625–30 ng/mL (r = 0.998) with an analytical detection limit of ~0.300 ng/mL (12 pg on column). Relative recoveries for all analytes were approximately ≥90% and the analytical run time was <10 min. The described method utilized minimal sample preparation procedures and was optimized to provide the sensitivity limits required for simultaneous monoamine and metabolite analysis in small, discrete brain tissue samples.     

Determination of cycloserine in microdialysis samples using liquid chromatography–tandem mass spectrometry with benzoyl chloride derivatization

A new method for the analysis of cycloserine (4‐amino‐3‐isoxazolidinone, CYC) in rat microdialysis samples has been developed. This method consists of derivatizing the CYC with benzoyl chloride, which transforms primary amines into highly stable derivatives. An attractive feature of this method was that the derivatization reaction is straightforward and can be completed within 10 min. The formed derivative, in contrast to the non‐derivatized analyte, exhibited increased chromatographic retention and decreased matrix effects resulting from the co‐elution of other components using reversed‐phase liquid chromatography and on‐line switching. Detection on a quadrupole–linear ion trap mass spectrometer (AB3200 Q‐Trap) was performed using electrospray tandem mass spectrometry in multiple reaction monitoring mode. Various derivatization parameters were optimized in order to improve chromatographic separation and minimize ion suppression. In particular, the benzoylation reaction was improved to enhance the reproducibility and sensitivity of the chromatographic method. The transition m/z 207.1 → 105.1 was acquired to monitor the CYC derivatization products. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. A good linearity over the selected range (r > 0.99, range = 22–2200 mg/L), as well as accuracy and precision within ±7% of the target values, was obtained. The assay described herein was successfully applied to quantitatively measure CYC in the lung and blood of anesthetized rats.     

Chromatographic determination of biogenic amines in wines after treatment with ionic liquids as novel media

Room temperature ionic liquids (RTIL), 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([C₄MIM][PF₆]), were used as the novel media for derivatization, extraction and preconcentration of biogenic amines (BAs) in wines. Six BAs, tryptamine (Try), phenylethylamine (Phe), putrescine (Put), cadaverine (Cad), tyramine (Tyr) and spermine (Spe) were selected as the model compounds and dansyl chloride (Dns‐Cl) as the derivatizing reagent. The derivatizations of amines were conducted in water‐IL two‐phase system, in which the partition property of related substance in the IL was investigated and the mechanism ( http://www.iciba.com/mechanism/ ) of derivation and extraction of amines was discussed. The influencing factors, including sample volume, derivatizing reagent concentration, pH value and ultrasound reaction time, were optimized. The Dns‐amines were separated by high‐performance liquid chromatography (HPLC), and detected with UVD at 254 nm. For each of the tested amines, a good linearity was obtained in the concentration range of 0.1–20 mg/L with the correlation coefficient (R) of 0.9814–0.9930. The limits of detection reached μg/L level and the relative standard deviations (RSD, n=3) were between 3.2 and 8.1%. Satisfactory recovery for each BA was obtained, ranging from 82.3 to 114.0%. The developed method was successfully applied to determine six BAs in red wines and Chinese yellow wine.     

Pharmacokinetic properties of wogonin and its herb–drug interactions with docetaxel in rats with mammary tumors

Docetaxel, frequently used for the treatment of breast cancer, is mainly metabolized via hepatic cytochrome P450 (CYP) 3A in humans and is also a substrate of P‐glycoprotein (P‐gp). Wogonin has been shown to be able to modulate the activities of CYPs and P‐gp, and it could serve as an adjuvant chemotherapeutic agent. However, the impacts of co‐administration of wogonin and docetaxel on their pharmacokinetics have not been studied because of a lack of an analytical method for their simultaneous measurement. In the present study, we established an HPLC–MS/MS method for simultaneous measurement of wogonin and docetaxel in rat plasma, and it was then utilized to explore the pharmacokinetics of wogonin and the herb–drug interactions between wogonin and docetaxel after their combined administration in rats with mammary tumors. The rats received 10, 20 and 40 mg/kg wogonin via oral administration, with or without docetaxel intravenously administered at 10 mg/kg, and the plasma concentrations of wogonin and docetaxel were measured using the established and validated HPLC–MS/MS method. The C_max and AUC_0–t of wogonin were proportionally increased in the dose range from 10 to 40 mg/kg, suggesting a linear pharmacokinetics of wogonin. Moreover, the C_max and AUC_0–t of docetaxel and the AUC_0–t of wogonin were increased after co‐administration (p < 0.05), indicating increased in vivo exposures of both wogonin and docetaxel, which might lead to an increase in not only therapeutic but also toxic effects. Thus the alterations of pharmacokinetics should be taken into consideration when wogonin and docetaxel are co‐administered.     

Biotransformation of p‐Coumaric Acid (=(2E)‐3‐(4‐Hydroxyphenyl)prop‐2‐enoic Acid) by Momordica charantia Peroxidase

LC/MS³‐Guided biotransformation of p‐coumaric acid (=(2E)‐3‐(4‐hydroxyphenyl)prop‐2‐enoic acid; CA) with H₂O₂/Momordica charantia peroxidase at pH 5.0 and 45° in the presence of acetone has resulted in the isolation of three CA trimers, triCA1 ( 1 ), triCA2 (trans‐ 2 ), and triCA3 (cis‐ 2 ), and seven CA dimers, diCA1–diCA7, i.e., 3 – 9 , among which seven (triCA1–triCA3 and diCA1–diCA4) are new compounds and three (diCA5–diCA7) are known compounds. The structures were established by 2D‐NMR such as HSQC, HMBC, and NOESY measurements. The possible mechanism for the formation of the products is also discussed (Schemes 1–3). This is the first time that the biotansformation of p‐coumaric acid catalyzed by peroxidase in vitro was achieved. Compounds triCA3 (cis‐ 2 ), diCA1 ( 3 ), diCA5 ( 7 ), and diCA7 ( 9 ) exhibit a stronger antioxidative activity than the parent CA.     

In situ derivatization–liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl‐4‐hydroxyproline by liquid chromatography–tandem mass spectrometry

Polar analytes that possess protic functional groups have often been treated with alkyl chloroformates to decrease their polarity and increase their volatility prior to gas chromatography–mass spectrometry analysis. This derivatization reaction has two distinct advantages. It proceeds smoothly in aqueous media, and the desired reaction products are efficiently separated from interfering ionic components by their extraction into a water‐immiscible organic phase. In the present work, the derivatization–liquid liquid sample preparation was examined in detail for analysis of a potential urinary dipeptide biomarker l ‐prolyl‐4‐l ‐hydroxyproline (PHP) by downstream liquid chromatography coupled to electrospray mass spectrometry. PHP was treated with a series of alkyl and fluoroalkyl chloroformates in aqueous media, and the detected reaction products were investigated. Smooth conversion of PHP into the N‐isobutyloxycarbonyl isobutyl ester was accomplished by the coupled action of isobutanol, isobutyl chloroformate and the pyridine catalyst. This derivative afforded a highest detector response from all the derivatized forms examined, including the nonderivatized PHP. A simple isocratic elution on a common RP‐C18 HPLC column coupled with tandem mass spectrometry, and use of the synthesized heptadeuterated analog (D7‐PHP) as an internal standard, enabled validation of the method and determination of PHP in human urine in less than 5 min. The in situ derivatization–liquid liquid extraction has thus been demonstrated to be a useful sample preparation strategy for the analysis of polar metabolites by liquid chromatography–tandem mass spectrometry in the complex urine matrix. Copyright © 2012 John Wiley & Sons, Ltd.