Quantitative method for determination of amphetamine in plasma using negative ion chemical ionisation GC-MS of o-(pentafluorobenzyloxycarbonyl)-benzoyl derivatives

The use of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)-benzoyl chloride is described for the quantitative determination of amphetamine in plasma. Amphetamine can be quantitatively measured down to 49 pg/mL plasma using only 250 μL of sample due to the extraordinary sensitivity of the derivatives under negative ion chemical ionisation mass spectrometry. Plasma samples were made alkaline with carbonate buffer and treated with n-hexane and reagent solution for 20 min, which, after concentration was measured by negative ion chemical ionisation gas chromatography-mass spectrometry. The method is rapid as extraction and derivatisation occur in one single step. [(2) H(5) ]-Amphetamine was used as an internal standard. Validation data are given to demonstrate the usefulness of the assay, including specificity, linearity, accuracy and precision, benchtop stability, freeze-thaw stability, autosampler stability, aliquot analysis and prospective analytical batch size accuracy.     

Quantitative determination of amphetamine in plasma using negative ion chemical ionization GC‐MS of o‐(pentafluorobenzyl‐oxycarbonyl)‐2,3,4,5‐tetrafluorobenzoyl derivatives

Quantitative determination of amphetamine in plasma by the use of a novel electrophoric derivatization reagent, o‐(pentafluorobenzyloxycarbonyl)‐2,3,4,5‐tetrafluorobenzoyl chloride is described. Amphetamine can be quantitatively measured down to 49 pg/mL plasma using only 250 μL of sample due to the extraordinary sensitivity of the derivatives under negative ion chemical ionization MS. Plasma samples were made alkaline with carbonate buffer and treated with n‐hexane and reagent solution for 20 min, which, after concentration was measured by negative ion chemical ionization GC‐MS. The method is rapid as extraction and derivatization occur in one single step. [²H₅]‐Amphetamine was used as an internal standard. Validation data are given to demonstrate the usefulness of the assay, including specificity, linearity, accuracy and precision, benchtop stability, freeze–thaw stability, autosampler stability, aliquot analysis, and prospective analytical batch size accuracy.     

GC-MS determination of parabens,triclosan and methyl triclosan in water by in situ derivatisation and stir-bar sorptive extraction

Stir-bar sorptive extraction in combination with an in situ derivatisation reaction and thermal desorption–gas chromatography–mass spectrometry was successfully applied to determine parabens (methylparaben, isopropylparaben, n-propylparaben, butylparaben and benzylparaben), triclosan and methyltriclosan in water samples. This approach improves both the extraction efficiency and the sensitivity in the GC in a simple way since the derivatisation reaction occurs at the same time as the extraction procedure. The in situ derivatisation reaction was carried out with acetic anhydride under alkaline conditions. Thermal desorption parameters (cryofocusing temperature, desorption flow, desorption time, desorption temperature) were optimised using a Box–Behnken experimental design. All the analytes gave recoveries higher than 79%, except methylparaben (22%). The method afforded detection limits between 0.64 and 4.12 ng/L, with good reproducibility and accuracy values. The feasibility of the method for the determination of analytes in water samples was checked in tap water and untreated and treated wastewater.     

Analytical method for assessing potential dermal exposure to pesticides of a non-agricultural occupationally exposed population

To measure dermal exposure of a non-agricultural occupationally exposed population to pesticides, a new method has been developed for analysis of 13 pesticides from different classes (fungicides, herbicides, insecticides) on dermal patches. The method includes extraction of the patches and analysis of the pesticides by GC–MS and/or HPLC–fluorescence. Water-soluble pesticides (glyphosate and glufosinate) on patches were ultrasonically extracted twice with ultra-pure water for 10 min and analysed by HPLC–fluorescence after derivatisation with FMOC. Organic-soluble pesticides (bifenthrin, cyprodinil, difufenicanil, fludioxonil, oxadiazon, pyriproxyfen, clopyralid, 2,4-D, fluroxypyr, 2,4-MCPA, and triclopyr) were extracted ultrasonically twice for 10 min with 70:30 dichloromethane–acetonitrile and analysed by GC–MS directly or after derivatisation with N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide. Detection limits varied between 3 and 4 μg L⁻¹ for water-soluble pesticides and between 1 and 10 μg L⁻¹ for organic-soluble pesticides.     

Determination of butoxyacetic acid (biomarker of ethylene glycol monobutyl ether exposure) in human urine candidate reference material

Ethylene glycol monobutyl ether (EGBE), an industrial solvent, is absorbed by the body not only by inhalation but also by dermal absorption (liquid or vapour). EGBE is metabolized to butoxyacetic acid (BAA). Pooled freeze-dried urine candidate reference material (RM) was prepared from urine obtained from persons occupationally exposed to EGBE. This material has the advantage of containing butoxyacetic acid in both the free and conjugated (glutamine and glycine) forms, as found in native urine. In all GC method modifications used, acid hydrolysis was used to release BAA from its conjugated form. The amount of butoxyacetic acid in homogeneity and stability testing was measured by GC after derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Detection was by MS in EI mode, in the authors’ laboratory. For interlaboratory comparison of the reference material GC methods with MS, FID, and ECD were used. Different extraction solvents (dichloromethane–isopropanol 2:1, ethyl acetate, or dichloromethane) and derivatisation reagents (trimethylsilyldiazomethane, N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide) were used. Using ANOVA (at the statistical level α = 0.05) no changes were found in the concentration of butoxyacetic acid during fifteen month isochronous stability testing, or in homogeneity testing. The uncertainty contributions were u _h = 8.8 mg L⁻¹ and u _s = 6.5 mg L⁻¹. The concentration of butoxyacetic acid in freeze-dried urine RM was evaluated from the results of eight laboratory data sets within an interlaboratory comparison by use of the interactive statistical software IPECA. The contribution to total uncertainty derived from interlaboratory comparison was u _i = 12.7 mg L⁻¹. The reference value (c = 273 ± 33 mg L⁻¹) is an unweighted arithmetic average of accepted results. The value is traceable to the pure butoxyacetic acid (98% w/w; Acros Organic #257760010) used as calibrant. The uncertainty given is combined expanded uncertainty derived from the results from interlaboratory comparison, and from homogeneity and stability tests (k = 2). The reference material will be used to verify method performance in the biological monitoring of occupational exposure to EGBE.     

Simple and rapid determination of zaltoprofen in human plasma by manual‐shaking‐assisted dispersive liquid–liquid microextraction followed by liquid chromatography with ultraviolet detection

A readily applicable method was developed to determine the concentration level of zaltoprofen, a non‐steroidal antiinflammatory drug from the propionic acid family, in human plasma. This method is based on manual‐shaking‐assisted dispersive liquid–liquid microextraction coupled with liquid chromatography with ultraviolet detection. Factors affecting the extraction efficiency were screened and optimized by experimental design using fractional factorial and central composite designs, respectively. Optimal conditions were: 220 μL of C₂H₄Cl₂ (extraction solvent), 5 mL of 3.75% w/v NaCl aqueous solution at pH 2.0, and manual shaking for 13 s (65 times). The resulting extraction method yielded a reasonable enrichment factor of 18.0 (±0.6, n  = 3) and extraction recovery of 86.0% (±3.3%, n  = 3). The established method was validated for selectivity, linearity, precision, accuracy, matrix effect, recovery, dilution integrity, and stability, and it met the acceptable criteria for all of the tested parameters. Specifically, the method was linear in the range of 0.16–50.0 mg/L, precise (< 8.8% RSD), accurate (–7.5–5.6% deviation), and showed negligible matrix effects (96.1–106.4%) with high absolute recovery (94.5–97.7%). Compared with previous methods involving labor‐intensive liquid–liquid extraction or non‐specific protein precipitation, our method allows the simple, rapid, and efficient determination of zaltoprofen using the most affordable analytical instrument, liquid chromatography with ultraviolet detection.     

Solid-phase micro-extraction procedure for the determination of 1,3-dichloro-2-propanol in water by on-fibre derivatisation with bis(trimethylsilyl)trifluoroacetamide

The headspace solid-phase micro-extraction technique with on-fibre derivatisation followed by gas chromatography-tandem mass spectrometry has been evaluated for the analysis of 1,3-dichloro-2-propanol in water. An asymmetric factorial design has been performed to study the influence of five experimental factors: extraction time and temperature, derivatisation time and temperature and pH. The best extraction performance is achieved in the headspace mode, with 5 mL stirred water samples (pH 4) containing 1.3 g of NaCl, equilibrated for 30 min at 25 °C, using divinylbenzene-carboxen-polydimethylsiloxane as the fibre coating. On-fibre derivatisation has been used for the first time with 50 μL of bis(trimethylsilyl)trifluoroacetamide at 25 °C during 15 min, leading to effective yields. The proposed method provides high sensitivity, good linearity and repeatability (relative standard deviation of 5.1% for 10 ng mL⁻¹ and n = 5). The limits of detection and quantification were 0.4 and 1.4 ng mL⁻¹, respectively. Analytical recoveries obtained for different water samples were approx. 100%.     

Optimisation of the on-fibre derivatisation of volatile fatty acids in the simultaneous determination together with phenols and indoles in cow slurries

The on-fibre derivatisation of volatile fatty acids (VFAs) using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was optimised in the simultaneous determination of VFAs together with phenols and indoles by headspace solid-phase microextraction (SPME)–gas chromatography–mass spectrometry. Firstly, the nature of the SPME fibre was optimised and four different fibres were studied (100 μm polydimethylsiloxane, 85 μm Carboxen/polydimethylsiloxane, 5/30 μm divinylbenzene/Carboxen/polydimethylsiloxane and 85 μm polyacrylate). The optimum fibre (50/30 μm divinylbenzene/Carboxen/polydimethylsiloxane) was used to study the exposure time of the fibre to the derivatisation agent and the desorption time and temperature. Firstly, a factorial design was built but since the three variables had a significant effect, a central composite design was used to build the response surfaces. The best signals were obtained after the exposure of the fibre in the headspace of the MTBSTFA derivatisation reagent for 1 h and desorption at 300 °C for 9 min. The determination of underivatised phenols and indoles was not affected by the presence of the derivatisation reagent in the fibre.     

Dried plasma spots as an alternative sample collection technique for the quantitative LC-MS/MS determination of gabapentin

Dried plasma spots were employed as an alternative sample collection technique for the quantitative determination of gabapentin in human plasma, using an automated liquid chromatography tandem mass spectrometry method. After the methanolic extraction of single plasma, 1/8-in. disks which contained only 1.98 μL plasma volume, were placed in 96-well format plates, then gabapentin and its internal standard, 4-aminocyclohexanecarboxylic acid, were derivatized with n-butanol/HCl (3 M) and detected as butyl esters. The assay exhibited excellent linearity over the concentration range of 40.0–10.0 × 10³ ng/mL, which is suitable for the determination of gabapentin after per os administration of a single tablet. Variations in intra- and inter-assay accuracy and precision were within internationally accepted criteria. Homogeneity of plasma spots was proven, whilst butyl ester stability for both analytes was estimated and found very satisfactory. The quantitative analysis of Gabapentin with dried plasma spot specimens seems to be a prominent and advantageous technique, especially when applied to pharmacokinetic studies, where plasma sampling procedure becomes rapid and required plasma volumes negligible.     

A versatile electrophoric derivatisation reagent for negative ion chemical ionisation mass spectrometry: <Emphasis Type="Italic">o</Emphasis>-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride

The synthesis of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride, is described. The reagent was tested against selected primary and secondary amino compounds, as well as phenolic and aliphatic hydroxyl compounds as analytical targets. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. Since the reagent bears a pentafluorobenzyl ester group, resulting negative ion chemical ionisation mass spectra were expectedly dominated by dissociative resonance electron capture typically observed with these compounds, additionally showing neutral loss of carbon dioxide and ammonia (in the case of primary amines). The reagent is suitable for detecting the target compounds with high sensitivity, as exemplified for the analysis of amphetamine and methylphenidate from human plasma where chromatographic background is drastically reduced by a shift in detected m/z and retention time and lower limits of quantification at 7.8 pg/mL (amphetamine) and 4.5 pg/mL (methylphenidate) can be obtained. The choice of two or three target quantification masses allows selective detection and adjustment of lowest background interference. No carryover effect was observed for the derivatives of amphetamine and methylphenidate.     

Determination of ethylphenols in wine by in situ derivatisation and headspace solid-phase microextraction–gas chromatography–mass spectrometry

Contamination by Brettanomyces is a frequent problem in many wineries that has a dramatic effect on wine aroma and hence its quality. The yeast Brettanomyces/Dekkera is involved in the formation of three important volatile ethylphenols—4-ethylphenol, 4-ethylguaiacol and 4-ethylcatechol—that transmit an unpleasant aroma to wine that has often been described as ‘medicinal’, ‘stable’ or ‘leather’. This study proposes an in situ derivatisation and headspace solid-phase microextraction– gas chromatography coupled to mass spectrometry method to determine the three ethylphenols in red Brettanomyces-tainted wines. The most important variables involved in the derivatisation (acetic anhydride and base concentration) and the extraction (extraction temperature and salt addition) processes were optimised by experimental design. The optimal conditions using 4 mL of wine in 20-mL sealed vials were 35 μL of acetic anhydride per millilitre of wine, 1 mL of 5.5% potassium carbonate solution and 0.9 g of sodium chloride and the extraction was performed with a divinylbenzene–carboxen–poly(dimethylsiloxane) fibre at 70 °C for 70 min. Then, the performance characteristics were established using wine samples spiked with the ethylphenols. For all compounds, the detection limits were below the odour threshold reported in the literature and they were between 2 and 17 μg L⁻¹ for 4-ethylguaiacol and 4-ethylphenol, respectively. Intermediate precision (as relative standard deviation) was acceptable, with values ranging from 0.3 to 12.1%. Finally, the method was applied in the analysis of aged Brettanomyces-tainted wines.     

Quantitative estimation of riluzole in human plasma by LC-ESI-MS/MS and its application to a bioequivalence study

A novel simple, sensitive, selective, and rapid high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for quantification of riluzole in human plasma. The chromatography was performed by using a Zorbax-SB-C18 (4.6 × 75 mm, 3.5 μm) column , isocratic mobile phase 0.1% formic acid/acetonitrile (10:90 v/v), and an isotope-labeled internal standard (IS), [¹³C,¹⁵N₂]riluzole. The extraction of drug and internal standard was performed by liquid–liquid extraction and analyzed by MS in the multiple reaction monitoring (MRM) mode using the respective [M+H]⁺ ions, m/z 235.0/165.9 for riluzole and m/z 238.1/169.0 for the IS. The calibration curve was linear over the concentration range 0.5–500.0 ng/ml for riluzole in human plasma. The limit of quantification (LOQ) was demonstrated at 0.5 ng/ml. The within-batch and between-batch precision were 0.6–2.3% and 1.4–5.7%, and accuracy was 97.1–101.1% and 98.8–101.2% for riluzole respectively. Drug and IS were eluted within 3.0 min. The validated method was successfully applied in a bioequivalence study of riluzole in human plasma.     

Direct spectrophotometric determination of rare earths in organic extracts with chlorophosphonazo III

The highly sensitive spectrophotometric determination of rare earths after extraction into di(2-ethylhexyl)phosphoric acid and trioctylamine is described. A sample of extract is diluted with n-butanol, the solution of chlorophosphonazo III in n-butanol is added, and the solution is made up to defined volume. Absorbance is measured at 660–665 nm against a reagent blank. The method is suitable for the study of the extraction of individual rare earths by different organic extrahents.     

Rapid solid‐phase extraction coupled with GC–MS method for the determination of venlafaxine in rat plasma: Application to the drug–drug pharmacokinetic interaction study of venlafaxine combined with fluoxetine

A rapid and sensitive gas chromatography with mass spectrometry method for the determination of venlafaxine in rat plasma has been developed and applied to a drug–drug interaction study of fluoxetine on pharmacokinetics of venlafaxine in rats. Rat plasma was spiked with 2% aqueous ammonia before subjected to preactivated C₁₈ solid‐phase extraction columns and eluted with methanol. No endogenous interferences were observed under optimal condition. The calibration curve was linear (R ² = 0.9994) in the range of 10–1000 ng/mL. The quantification limit of venlafaxine in rat plasma was 10 ng/mL. The accuracy was in the range of 85–110%, and the extraction recovery was no less than 50%. Both the intra‐ and interday precision were 5.0–10.7%. The concentration–time curve showed that plasma concentrations of the coadministration group (group B) were higher than that of single dose group (group A). Both values of C _max (0.069 mg/L) and AUC_0→∞ (0.291 mg h/L) in group B were statistically greater than that of C _max (0.046 mg/L) and AUC_0→∞ (0.181 mg·h/L) in group A (P  < 0.05). The results indicated that a significant effect of fluoxetine was shown on the pharmacokinetics of venlafaxine, suggesting that drug–drug interactions are of concern for the treatment of depression with the combined use of venlafaxine and fluoxetine.     

A fluorous tag-bound fluorescence derivatization reagent, F-trap pyrene, for reagent peak-free HPLC analysis of aliphatic amines

We have developed a novel pre-column fluorescence derivatization reagent for amines, F-trap pyrene. This reagent comprises a fluorescent pyrene moiety, an amine-reactive Marshall linker, and a fluorophilic perfluoroalkyl group known as fluorous tag. When the reagent reacts with aliphatic amines and amino acids to give fluorescent derivatives, the fluorous tag in the reagent is eliminated simultaneously. Therefore, excess unreacted reagents in the derivatization reaction solution still have the fluorous tag and could be removed by fluorous solid-phase extraction selectively before high-performance liquid chromatography (HPLC) analysis. By using this reagent, 13 kinds of aliphatic amine (C₂–C₁₆) derivatives can be separated within 40 min by reversed-phase HPLC with gradient elution. In this chromatogram, unreacted reagents peak at around 28 min, greatly decrease after fluorous solid-phase extraction, and do not interfere with the quantification of each amine. The detection limits (S/N = 3) for examined aliphatic amines are 3.6–25 fmol per 20 μL injection. We have also applied this reagent successfully to the amino acid analysis.

Optimisation of stir bar sorptive extraction and in-tube derivatisation-thermal desorption-gas chromatography-mass spectrometry for the determination of several endocrine disruptor compounds in environmental water samples

The analysis of organic pollutants in environmental water samples requires a pre-concentration step. Pre-concentration techniques such as stir bar sorptive extraction (SBSE) have gained popularity since they minimise the use of toxic organic solvents and can be considered as green analytical techniques. Similar to other pre-concentration techniques, one of the problems when SBSE is used is the matrix effect, which often occurs during the analysis of environmental water samples such as estuarine or wastewater samples. The present work studied the matrix effect during SBSE coupled to in-tube derivatisation–thermal desorption (TD)–gas chromatography–mass spectrometry for the determination of several endocrine disruptor compounds, such as alkylphenols, bisphenol A, estrogens and sterols, in environmental water samples, after optimisation of the major variables affecting the determination. Variables such as the addition of methanol or an inert salt to the donor phase, the extraction temperature, the volume of the donor phase, the stirring rate and the extraction time were studied during the SBSE optimisation. In the case of the in-tube derivatisation and TD step, the volume of the derivatisation reagent (N,O-bis(trimethylsilyl)triufloroacetamide with 1% of trimethylchlorosilane (BSTFA + 1% TMCS)) and the cryo-focusing temperature were fixed (2 μL and −50 °C, respectively) according to a consensus between maximum signal and optimal operation conditions. Good apparent recovery values (78–124%) were obtained for most of the analytes in Milli-Q water, except for 4-tert-octylphenol (4-tOP), which showed apparent recovery values exceeding 100%. Precision (n = 4) was in the 2–27%, and method detection limits were in the low nanogrammes per litre level for most of the analytes studied. The matrix effect was studied using two different approaches. On the one hand, Milli-Q water samples were spiked with humic acids, and apparent recovery values were studied with and without correction with the corresponding deuterated analogue. On the other hand, estuarine water and wastewater samples were spiked with known concentrations of target analytes, and apparent recoveries were studied as explained above. In general, the matrix effect could be corrected with the use of deuterated analogues, except for 4-tOP and nonylphenols for which [²H₄]-n-nonylphenol did not provide good corrections.     

Development of a liquid chromatography-tandem mass spectrometry method for plasma-free metanephrines with ion-pairing turbulent flow online extraction

Liquid chromatography–tandem mass spectrometry has become the preferred technology to measure unconjugated metanephrine and normetanephrine in plasma because of its high sensitivity and specificity over immunoassay and gas chromatography–mass spectrometry. In our earlier study, plasma metanephrines were extracted with offline ion-pairing solid-phase extraction and quantified by liquid chromatography–tandem mass spectrometry with porous graphitic carbon column based chromatography. In this study, we aim to automate the sample preparation with turbulent flow online extraction technology and maintain or improve the analytical performance previously achieved from the offline approach. The online extraction was done with a mixed-mode cation exchange turbulent flow chromatography column assisted with ion-pairing reagent and porous graphitic column was used for chromatographic separation. The total online extraction and analytical LC runtime was 12 min. This method was linear from 6.3 to 455.4 pg/mL for metanephrine; 12.6 to 954.5 pg/mL for normetanephrine with an accuracy of 80.6% to 93.5% and 80.9% to 101.7%, respectively. The lower limit of quantitation was 6.3 pg/mL for metanephrine and 12.6 pg/mL for normetanephrine. Inter-assay and intra-assay precision for metanephrine and normetanephrine at low and high concentration levels ranged from 2.0% to 10.5%. In conclusion, we have developed a fast and sensitive automated online turbulent flow extraction method for the quantitative analysis of plasma metanephrines. Ion-pairing reagent was necessary for the success of this method.     

On-line determination of aliphatic amines in water using in-tube solid-phase microextraction-assisted derivatisation in in-valve mode for processing large sample volumes in LC

This paper describes a cost-effective procedure for the analysis of short-chain aliphatic amines in water samples using a solid-phase microextraction device. Analyte preconcentration and derivatisation were effected into a capillary column coated with 95% polydimethylsiloxane–5% polydiphenylsiloxane, which was used as the injection loop of a Rheodyne injection valve. The coating was previously loaded with the derivatisation reagent, 9-fluorenylmethyl chloroformate. A volume of 1 mL of samples was then drawn into the capillary column, and the extracted analytes were left to react on the capillary coating for 5 min. Next, the capillary column was cleaned by passing water. Finally, the injection valve was rotated, and the derivatives formed were dynamically desorbed and transferred to the analytical column into the mobile phase. Methylamine, ethylamine, propylamine, n-butylamine and n-pentylamine were selected as model compounds. Excellent sensitivity was achieved, being the limits of detection of 15–200 μg/L when using UV detection and of 0.1–0.4 μg/L by fluorescence.     

A sensitive liquid chromatographic-mass spectrometric method for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma

A liquid chromatographic–mass spectrometric (LC–MS) method has been developed and validated for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma. After addition of the internal standard, domperidone, plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Apollo C₁₈ column (250 mm × 4.6 mm, 5 μm), with methanol–0.01% formic acid water (60:40, v/v) as mobile phase, within a runtime of 12.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 1.0–500 ng mL⁻¹ for dehydroevodiamine and 2.0–1,000 ng mL⁻¹ for limonin, with lower limits of quantitation of 1.0 and 2.0 ng mL⁻¹, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, respectively, for both analytes, and the accuracy (relative error, RE, %) was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to a comparative pharmacokinetic study of dehydroevodiamine and limonin in rat plasma after oral administration of dehydroevodiamine, limonin, and an aqueous extract of Evodiae fructus. The results indicated there were obvious differences between the pharmacokinetic behavior after oral administration of an aqueous extract of Evodiae fructus compared with single substances.     

A method for quantifying the unstable and highly polar drug nafamostat mesilate in human plasma with optimized solid-phase extraction and ESI-MS detection: more accurate evaluation for pharmacokinetic study

An advanced quantification method was developed with solid-phase extraction (SPE) and mass spectrometry (MS) determination for nafamostat, an unstable and highly polar drug, in human plasma. For unstable drugs with an ester group, the main analytical challenge is how to avoid the ester hydrolysis, and strong acid or alkaline conditions should be excluded during sample preparation. Considering that, we developed a relatively mild method with SPE for sample preparation without strong acid and alkaline treatment, which was optimized with different pHs and salt concentrations in phosphate-buffered saline treatment. The results indicated that pH 5 gave the most efficient extraction and 0.1 M salt concentration enhanced the extraction the most, with a minor effect on MS monitoring. The extraction method effectively avoided drug hydrolysis and achieved good drug enrichment over 82.2%. The linear range of quantification was 1.25–160 ng mL⁻¹. The stability of the drug in sample treatment was fully validated according to the sample processing procedure, including the stability in fresh blood, mobile phase, plasma and acidic methanol, and the results indicated that the drug remained stable during the whole sample preparation. Compared with a previous isotope-labeling method, more accurate and specific quantification of plasma concentration was achieved with liquid chromatography–electrospray ionization MS determination. With use of our method, nafamostat mesilate pharmacokinetics in 30 Chinese healthy volunteers was investigated with three doses via intravenous-drip infusion. The pharmacokinetic parameters were also estimated and compared with those of Japanese volunteers (slightly lower plasma concentration and longer terminal elimination half-life for Chinese volunteers). The difference in the pharmacokinetics may be ascribed to the quantification method, because previous isotope labeling may have overestimated the parent drug.