超高效液相色谱-串联质谱法测定食品包装材料中全氟辛烷磺酸盐

建立了超高效液相色谱-串联质谱(UPLC- MS/MS)测定食品包装材料中全氟辛烷磺酸盐(PFOS)的方法.采用乙腈作为溶剂,加速溶剂提取法提取食品包装材料中的PFOS.色谱条件:ACQUITY UPLC BEH C₁₈色谱柱（1.7 μm,2.1 mm×50 mm）;柱温:30 ℃;流动相:乙腈/水,梯度洗脱;流速:0.2 mL/min;经UPLC分离后用多级反应监测(MRM)方式测定.用2个子离子的相对丰度定性, 外标法定量.PFOS在0.005～0.500 μg/mL范围内线性良好(R2=0.999),PFOS的回收率为90.0%～101.6%,相对标准偏差RSD为1.5%～3.5%.方法检出限为0.1 μg/m2(S/N≥3).     

市售虾中磺胺甲噁唑的LC-MS-MS定量分析

当前各国对食品安全都极为重视,我国以及发达国家相继制定了相关兽药用量的法规和标准,尽管中国已经加入到了WTO,但我国的畜禽产品出口还是遇到了很多困难,农药、兽药、饲料添加剂等非法滥用,造成药物残留而直接影响到人类的健康。如引发癌症,婴儿畸形,代谢紊乱以及抗药性等诸多问题。其中磺胺类兽药是一类受到严格限制使用的兽药,本文研究了市售虾中磺胺甲(口恶)唑定量分析方法,检出限绝对量可达到0.5pg,完全满足发达国家的最低检出规定的苛刻标准。     

Solid-phase extraction of 4(5)-methylimidazole (4MeI) and 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI) from foods and beverages with subsequent liquid chromatographic-electrospray mass spectrometric quantification

A method for the simultaneous determination of 4(5)-methylimidazole (4MeI) and 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI) was developed using SPE and HPLC/MS. Solid-phase extraction using SCX Disc cartridges was used for isolation of the analytes from liquid samples. The lower LOQwas 0.1 ng/mL for 4MeI and 0.2 ng/ mL for THI. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients >0.999. The CV for the intra- and inter-day precision was <5% (n = 6); the accuracy was in the range 98-103%. The recovery was > or = 97 and > or = 98% for THI and 4MeI, respectively. The method was used to determine THI and 4MeI in beverages, coffee, caramel colours and other samples.     

Liquid chromatography/tandem mass spectrometry for analysis of 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) and 1,2,5,6-tetrabromocyclooctane (TBCO)

Although the two flame retardants 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) and 1,2,5,6-tetrabromocyclooctane (TBCO) have been widely used, a selective instrumental method of analysis for these compounds has not been developed to date. In this study, we demonstrate the feasibility to utilize liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the separation and analysis of α- and β-TBCO and α-, β-, γ-, and δ-TBECH. Acetone was initially used in a tetrahedron solvent system for LC optimization. A simple isocratic elution allowed near-baseline separation of these compounds. Different ionization approaches and mechanisms were investigated. The mass spectrometric transition of [M + O(2)](-) => Br(-) (459.8 => 78.9) was a selective detection method for the target analytes. Good instrument detection limits (5 pg for γ-/δ-TBECH, 125 pg for α-/β-TBECH, and 30 pg for α-/β-TBCO with 2.0 μL injection) were obtained. Excellent linearity up to 50 ng/μL (R(2) >0.999) was also achieved. This method has been applied to environmental samples (surface water) for screening purposes with recoveries ranging from 76-92% (CV%: 5-8%). This method shows significant improvement over previous methods.     

Stereoselective determination of ginsenosides Rg3 and Rh2 epimers in rat plasma by LC‐MS/MS: Application to a pharmacokinetic study

We developed and validated an accurate and sensitive LC–MS/MS method for the simultaneous quantitation of ginsenoside Rg3 and Rh2 epimers (R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2) in rat plasma. Analytes were extracted from 0.1 mL aliquots of rat plasma by liquid–liquid extraction, using 2 mL of ethyl acetate. In this assay, dioscin (500 ng/mL) was used as an internal standard. Chromatographic separation was conducted using an Acclaim RSLC C₁₈ column (150 × 2.1 mm, 2.2 μm) at 40°C, with a gradient mobile phase consisting of 0.1% formic acid in distilled water and in acetonitrile, a flow rate of 0.35 mL/min, and a total run time of 20 min. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with negative electrospray ionization at m/z 783.4 → 161.1 for R‐Rg3 and S‐Rg3, m/z 621.3 → 161.1 for R‐Rh2 and S‐Rh2, and m/z 867.2 → 761.5 for the internal standard. For R‐Rg3 and S‐Rg3, the lower limit of quantification was 5 ng/mL, with a linear range up to 500 ng/mL; for R‐Rh2 and S‐Rh2, the lower limit of quantification was 150 ng/mL, with a linear range up to 6000 ng/mL. The coefficient of variation for assay precision was less than 10.5%, with an accuracy of 86.4–112%. No relevant cross‐talk or matrix effect was observed. The method was successfully applied to a pharmacokinetic study after oral administration of 400 mg/kg and 2000 mg/kg of BST204, a fermented ginseng extract, to rats. We found that the S epimers exhibited significantly higher plasma concentrations and area under curve values for both Rg3 and Rh2. This is the first report on the separation and simultaneous quantification of R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2 in rat plasma by LC‐MS/MS. The method should be useful in the clinical use of ginseng or its derivatives.     

在线液相-气相二维色谱高灵敏测定卷烟主流烟气中的4-（N-甲基亚硝胺基）-1-（3-吡啶基）-1-丁酮

建立了在线液相-气相二维色谱测定卷烟主流烟气中4-(N-甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)的方法。 NNK 的分析在在线凝胶气质联用仪上进行,采用自行装填的微型碱性氧化铝柱,并把仪器上的凝胶柱换成氧化铝柱,用于 NNK 的分析。捕集有主流烟气总粒相物的剑桥滤片用二氯甲烷提取,以 D4-NNK 为内标,提取液经微型氧化铝柱分离,含 NNK 的部位切割进入气相色谱,排干溶剂后启动气相色谱升温经毛细管柱进行分离,用质谱检测。本方法将烟气国标方法 NNK 测定中的氧化铝柱色谱净化和气相色谱-质谱分析在线连接起来,可不经样品前处理净化直接进样分析;每次进样可达40μL,是常规气相色谱-质谱分析最大进样量(2.0μL)的20倍,显著提高了分析灵敏度。方法线性范围达1.2~120 ng/ mL,相关系数为r=0.9998,回收率为93.9%~96.0%;检出限和定量限分别为0.25 ng/ mL 和0.9 ng/ mL,样品分析结果与中国烟草总公司企业标准方法进行对比,结果相符合。     

Coupling of acetonitrile deproteinization and salting-out extraction with acetonitrile stacking for biological sample clean-up and the enrichment of hydrophobic compounds (porphyrins) in capillary electrophoresis

A new sample pretreatment approach in CE was developed for concurrent biological sample clean-up and the concentration of hydrophobic compounds based on the combination of ACN deproteinization with salting-out extraction. Further enhancement in concentration detection sensitivity was achieved by coupling (offline) salting-out extraction with an online CE sample enrichment technique known as "ACN stacking". By optimizing the pH of salting-out extraction, a number of model compounds (hydrophobic porphyrins with clinical significances), i.e. zinc-protoporphyrin, protoporphyrin, and coproporphyrin (CP) III and I, can be efficiently extracted from the aqueous sample into a smaller volume organic solvent (ACN) phase and an enrichment factor of ca. 100 can be obtained. The pressure injection of the enriched ACN phase (containing ca.1% NaCl) into the CE capillary at 10% capillary volume resulted in additional concentration of the various hydrophobic porphyrins, allowing for a combined enrichment factor of ca.1000 to be obtained. Calibration curves obtained for the determination of a pair of positional isomers with significant diagnostic value, urinary CPIII and CPI, were found to be linear between 10-300 ng/mL (with R2 = 0.999), and LODs (absorbance detection at 400 nm) were ca. 0.8 ng/mL (1.1 nmol/L of CPIII or CPI). Based on a single salting-out extraction, intraday precisions (nine consecutive injections) for both CPIII and CPI (at spiked concentrations of 10-300 ng/mL into urine) in terms of migration time and peak area were found to be within the range of 0.2-0.5 and 0.8-2.9%, respectively.     

超高效液相色谱-三重四极杆质谱联用方法测定血浆和尿液中的α-龙葵碱、α-卡茄碱和茄啶

建立了超高效液相色谱-三重四极杆质谱联用方法,检测血浆和尿液中的α-龙葵碱、α-卡茄碱和茄啶。样品经2%(v/v,下同)甲酸水溶液等量稀释,再经混合型阳离子交换固相萃取柱(MCX SPE)净化,以0.1%甲酸乙腈溶液和含0.05%甲酸的5 mmol/L乙酸铵水溶液作为流动相进行梯度洗脱,在UPLC BEH C18色谱柱上实现分离,正离子电喷雾串联质谱多反应监测(ESI-MS/MS MRM)方式检测,基质匹配外标法定量。一次进样分析时间为5.5min。血浆和尿液中3种待测物的线性范围均为0.3~100 ng/mL,相关系数为0.997~0.999;样品的检出限为0.1 ng/mL,定量限为0.3 ng/mL;血浆和尿液中的平均加标回收率分别为82%~112%和96%~114%,相对标准偏差为4.0%~16%和2.7%~17%(n=6)。方法简单、准确、灵敏,适用于马铃薯中毒检测。     

Separation and preconcentration of trace amounts of gold(III) ions using modified multiwalled carbon nanotube sorbent prior to flame atomic absorption spectrometry determination

Multiwalled carbon nanotubes are attractive as sorbents for SPE because they can be used for enrichment of organic compounds and metal ions at trace levels. In this study, multiwalled carbon nanotubes were oxidized with concentrated HNO3, and then the oxidized multiwalled carbon nanotubes were modified with 5-(4'-dimethylamino-benzyliden)-rhodanine. The modified multiwalled carbon nanotubes were used as a solid sorbent for separation and preconcentration of trace amounts of Au(III) ions. The sorption of Au(III) ions was quantitative in the pH range of 2.0-5.0, whereas quantitative desorption occurred instantaneously with 5.0 mL 2.0 M Na2S2O3. The eluted solution was aspirated directly into the flame for atomic absorption spectrometry. The proposed method resulted in an enrichment factor of 94. The RSD of the method was +/- 1.11% (n=10, 2.0 microg/mL) and the LOD was 0.15 ng/mL. The calibration curve for Au(III) was linear between 0.53 ng/mL and 36.0 microg/mL in the initial solution, with an R2 value of 0.9999. The sorbent capacity of the modified multiwalled carbon nanotubes was 7.3 mg Au(III)/g sorbent. The influences of the experimental parameters, including sample pH, sample flow rate, eluent volume and flow rate, sample volume, and interference of some ions on the recoveries of the Au ions, were investigated. The proposed method was applied for preconcentration and determination of Au in different samples.     

A rapid and sensitive liquid chromatography/tandem mass spectrometry method for determination of docetaxel in human plasma

A novel, rapid and sensitive isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for quantification of docetaxel in human plasma with paclitaxel as internal standard. The high sensitivity and specificity of MS/MS detection enabled the use of a small volume of plasma (0.05 mL) and a simple liquid-liquid extraction procedure. Furthermore, a very short run-time (3 min) fulfilled the need for monitoring plasma levels of docetaxel from large-scale clinical studies. The calibration curve for docetaxel was linear over the range 5-1000 ng/mL with coefficients of correlation >0.999 using only 0.05 mL plasma. The intra- and inter-day precisions (CV) of analysis were <7%, and accuracy ranged from 96 to 110%. The applicability of the method was demonstrated in a pharmacokinetic study of a 1-h infusion of docetaxel with dosages of 75 mg/m(2). Possible conjugated metabolites of docetaxel were not detected in patients' samples.     

Determination of proanthocyanidins in fresh grapes and grape products using liquid chromatography with mass spectrometric detection

Fresh grapes and grape products, such as grape wine and grape juice, were analyzed for proanthocyanidins (PACs) using liquid chromatography with electrospray ionization mass spectrometric (MS) detection. PACs were successfully separated and analyzed on the basis of their protonated molecules, allowing the identification of PACs in different degrees of polymerization from monomers to oligomers (up to 7 units), and in various isomeric forms. Using reversed-phase high-performance liquid chromatography (HPLC) combined with MS detection, the PAC monomers, (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), and (-)-epicatechin gallate (ECG), were successfully quantified using selected ion monitoring (SIM) mode. Standard curves were fitted for each PAC ranging from 43.8 to 5600 ng/mL for C, from 42.2 to 5400 ng/mL for EC, from 36.7 to 4700 ng/mL for CG, and from 39.8 to 5100 ng/mL for ECG. Good linearity (r2>0.999) was achieved for each analyte. The accuracy and precision (RSD) were within 10% (n=8) at the limit of detection. This method allows direct quantification of monomeric PACs in fresh grapes and grape-derived products. Additionally, flow injection analysis (FIA) was applied to estimate the concentration levels of PAC oligomers by comparing their FIA-MS peak areas, which were well correlated (r2=0.936) to the total concentrations of PAC monomers.     

Liquid chromatography/tandem mass spectrometry for pharmacokinetic studies of 20(R)-ginsenoside Rg3 in dog

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-ginsenoside Rg3 in dog. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. Dioscin was used as the internal standard. The method had a lower limit of quantitation of 0.5 ng/mL for Rg3 in 200 microL of plasma or 2 ng/mL in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of Rg3 in plasma. The intra- and inter-day precisions were measured to be below 8% and accuracy between -1.5 and 1.4% for all quality control samples. This quantitation method was successfully applied to pharmacokinetic studies of Rg3 after both an oral and an intravenous administration to beagle dogs. No Rh2 and protopanaxadiol were detected in plasma.     

Quantification of chlordesmethyldiazepam by liquid chromatography–tandem mass spectrometry: application to a cloxazolam bioequivalence study

A rapid, sensitive and specific LC‐MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid‐liquid extraction (diethyl‐ether/hexane, 80/20, v/v) procedure. The LC‐MS/MS method on a RP‐C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5–50 ng/mL (R > 0.999). The between‐run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification—LLOQ (0.500 ng/mL). The between‐run accuracies were 0.1, –1.5, –2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam—test, Eurofarma Lab. Ltda and Olcadil®— reference, Novartis Biociências S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUC0‐inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of acyclovir in horse plasma and body fluids by high-performance liquid chromatography combined with fluorescence detection and heated electrospray ionization tandem mass spectrometry

Two methods are presented for the determination of 'respectively' the plasma protein unbound and total concentration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon filter was performed. Ganciclovir was used as an internal standard. Analysis was carried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL(-1) for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5 ng mL(-1) and at 2 ng mL(-1) for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir.     

电感耦合等离子体质谱法测定铁基体中稀土元素Nd和Dy

采用电感耦合等离子体质谱法测定铁基体中的稀土元素Nd和Dy,选择187Re作为内标以校正仪器的信号漂移,Nd和Dy元素的浓度与信号强度线性关系良好,相关系数(r2)分别为0.9998和0.9997,该方法测定Nd和Dy元素的检出限分别为0.02 ng/mL和0.006 ng/mL,平均回收率分别为99.5％和97.4％,测定结果的相对标准偏差分别为3.5％和4.3％.     

Liquid chromatography high resolution mass spectrometry for the determination of baclofen and its metabolites in plasma: Application to therapeutic drug monitoring

Baclofen is used to manage alcohol dependence. This study describes a simple method using liquid chromatography coupled to high‐resolution mass spectrometry (LC‐HR‐MS) developed in plasma samples. This method was optimized to allow quantification of baclofen and determination of metabolic ratio of its metabolites, an oxidative deaminated metabolite of baclofen (M1) and its glucuronide form (M2). The LC‐HR‐MS method on Exactive® apparatus is a newly developed method with all the advantages of high resolution in full‐scan mode for the quantification of baclofen and detection of its metabolites in plasma. The present assay provides a protein precipitation method starting with 100 μL plasma giving a wide polynomial dynamic range (R ² > 0.999) between 10 and 2000 ng/mL and a lower limit of quantitation of 3 ng/mL for baclofen. Intra‐ and inter‐day precisions were <8.1% and accuracies were between 91.2 and 103.3% for baclofen. No matrix effect was observed. The assay was successfully applied to 36 patients following baclofen administration. Plasma concentrations of baclofen were determined between 12.2 and 1399.9 ng/mL and metabolic ratios were estimated between 0.4 and 81.8% for M1 metabolite and on the order of 0.3% for M2 in two samples.     

Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC-ESI-MS-MS

To investigate probable health benefits of flavonoids and stilbenes in red wine a new reversed-phase (RP) high-performance liquid-chromatographic (HPLC) method with enhanced separation efficiency and improved selectivity, sensitivity, and speed has been established for determination of the flavonoids quercetin, myricetin and kaempferol and the stilbenes cis- and trans-resveratrol, in a single run . UV-absorbance, fluorescence (FLD), and mass-spectrometric (MS) detection were also evaluated. UV-absorbance detection at 320 nm for stilbenes and 377 nm for flavonoids enables their determination up to the nanogram range with a linearity of R2>0.9999 (linear range 50 ng mL(-1)-50 microg mL(-1)). Calculated values of average recoveries were between 95 and 105% for all analytes. For resveratrol, fluorescence detection was highly selective and twice as sensitive as UV detection, and linearity was satisfactory (R2>0.9996; linear range see UV detection). For the detection of the hydrophilic glycosidic compounds piceid and rutin, which are coeluted with other hydrophilic ingredients, the validated RP HPLC system was coupled to a quadrupole ion-trap mass-spectrometer (MS) via an electrospray interface (ESI) with 25% ammonia solution as sheath liquid. MS detection was, highly linear (R2>0.9878; linear range 50 ng mL(-1)-50 microg mL(-1)) for all investigated analytes and the limits of detection were in the low nanogram range. Compared with UV detection MS detection resulted in a 200% increase in signal intensity for myricetin and 400% increases for quercetin and kaempferol, but equal signal intensity for resveratrol. Calculated values of average recoveries were 102% for myricetin and 79% for piceid. Collision induced dissociation (CID) was also used to obtain characteristic fragmentation fingerprints to facilitate qualitative and quantitative analysis even in complex matrices. Finally, this hyphenated HPLC-ESI-MS method was highly suitable and an essential improvement compared with UV- and fluorescence detection.     

Simultaneous determination of paclitaxel and a new P-glycoprotein inhibitor HM-30181 in rat plasma by liquid chromatography with tandem mass spectrometry

An LC-MS/MS method for the simultaneous determination of a new P-glycoprotein inhibitor 4-oxo-4H-chromene-2-carboxylic acid [2-(2-(4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxy-phenyl]-amide (HM-30181) and a P-glycoprotein substrate paclitaxel in rat plasma was developed to simultaneously evaluate the pharmacokinetics of paclitaxel and HM-30181 in the rats. HM-30181, paclitaxel, HM-30059 (internal standard (I.S.) for HM-30181), and docetaxel (I.S. for paclitaxel) were extracted from rat plasma with methyl-tert-butyl ether and analyzed on an Atlantis C18 column (5 microm, 2.1 x 100 mm) with the mobile phase of ACN/10 mM ammonium formate (75:25 v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring (MRM) mode. The standard curves for HM-30181 and paclitaxel in plasma were linear (r > 0.999) over the concentration range of 2.0-500 ng/mL with a weighting of 1/concentration2. The method showed a satisfactory sensitivity (2 ng/mL using 50 microL plasma), precision (CV: < or = 6.6%), accuracy (relative error: -6.3 to 2.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of HM-30181 and paclitaxel in rat plasma after oral-coadministration of paclitaxel and HM-30181 to male Sprague- Dawley rats.     

Determination of total and free concentrations of the enantiomers of methadone and its metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine) in human plasma by enantioselective liquid chromatography with mass spectrometric detection

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been validated for the determination of total and free plasma concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP, the primary metabolite of Met), using their respective deuterium-labeled compounds as internal standards [(R,S)-d3-Met and (R,S)-d3-EDDP]. For total drug determinations, 1 ml human plasma was extracted, using a cation-exchange solid-phase extraction cartridge; the eluate was evaporated, reconstituted in the mobile phase, and injected into the LC-MS system. The free fractions of Met and EDDP were determined, using 500 microl of plasma, which were placed in an ultrafiltration device and centrifuged at 2000 x g until 250 microl of filtrate was collected. The filtrate was extracted as described above and analyzed. Enantioselective separations were achieved using an alpha1-acid glycoprotein chiral stationary phase, a mobile phase composed of acetonitrile-ammonium acetate buffer [10 mM, pH 7.0] (18:82, v/v), a flow rate of 0.9 ml/min at 25 degrees C. Under these conditions, enantioselective separations were observed for Met (alpha = 1.30) and EDDP (alpha = 1.17) within 15 min. Met, EDDP, [2H3]-Met and [2H3]-EDDP were detected using selected ion monitoring at m/z 310.30, 278.20, 313.30, and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for Met in the range 1.0-300.0 ng/ml, and for EDDP from 0.1 to 25.0 ng/ml with correlation coefficients greater than 0.999, where the lower limit of quantification (LLOQ) was 1 ng/ml for Met and 0.1 ng/ml for EDDP. The relative standard deviation (R.S.D.) expressed as R.S.D. for the intra- and inter-day precision of the method were < 5.3% and the R.S.D. for accuracy was < 5.0%. The method was used to analyze plasma samples obtained from patients enrolled in a Met-maintenance program.     

Determination of 24,25-dihydroxyvitamin D(3) in human plasma using liquid chromatography-mass spectrometry after derivatization with a Cookson-type reagent

A practical LC-MS method for determination of (24R)-24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in human plasma has been developed using derivatization with a Cookson-type reagent, 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione (MBOTAD). The derivatization with MBOTAD significantly improved the ionization efficiency of the analyte with a detection limit of 18 fmol [equivalent to 7.5 pg of 24,25(OH)(2)D(3)] per injection. The method employed protein precipitation with acetonitrile, purification with OASIS HLB cartridge and silica gel column, derivatization with MBOTAD and atmospheric pressure chemical ionization MS detection. The mass spectrometer was operated in the positive-ion mode of mass chromatography and [26,26,26,27,27,27-(2)H(6)]-24,25(OH)(2)D(3) was used as an internal standard. The intra- and inter-assay coefficients of variation were below 3.4 and 2.5%, respectively, and the analytical recovery of 24,25(OH)(2)D(3) was quantitative. Assay linearity was obtained in the range of 0.05-1.2 ng per tube and the limit of quantitation was 0.23 ng/mL for a 0.3 mL plasma aliquot. The developed method was applied to plasma samples obtained from volunteers and gave satisfactory results.