Sensitive liquid chromatography/mass spectrometry assay for the quantification of azithromycin in human plasma

A simple, rapid, sensitive and specific liquid chromatography/electrospray ionization mass spectrometry method was developed and validated to quantify azithromycin in human plasma, using erythromycin as the internal standard (IS). A simple sample preparation method of protein precipitation with methanol was employed. Methanol, acetonitrile and water (12:30:58, v/v/v) were used as the isocratic mobile phase, with 0.1% formic acid and 0.1% ammonium acetate in water. Selected ion monitoring was specific for azithromycin and erythromycin. The assay was linear over the concentration range 4.69-600 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9994 to 0.9998. The intra- and inter-day precisions, calculated from quality control samples, were less than 8.24%. The method was employed in a pharmacokinetic study after oral administration of 500 mg azithromycin dispersible tablet to 20 healthy volunteers.     

A high-performance liquid chromatographic method for determination of scopolin in rat plasma: application to pharmacokinetic studies

An analytical method based on high-performance liquid chromatographic (HPLC) with ultraviolet (UV) detection was developed for determination of scopolin in rat plasma using aesculin as internal standard (IS). After protein precipitation of plasma sample with methanol, the supernatant was directly injected and analyzed. Chromatographic separation was achieved on a C18 column using methanol and distilled water (22:78, v/v) containing 0.2% (v/v) glacial acetic acid as mobile phase with a column temperature of 30 degrees C. The UV detector was set at 338 nm. The calibration curve was linear over the range of 0.105-13.125 microg/mL with a correlation coefficient of 0.9998. The retention times of aesculin and scopolin were 10.4 and 12.8 min, respectively. The recoveries for plasma samples of 0.105, 4.725 and 13.125 microg/mL were 91.08, 95.30 and 96.10%, respectively. The RSD of intra- and inter-day assay variations was less than 7.35%. The lower limit of detection was 0.03 microg/mL .This HPLC assay is a simple, sensitive and accurate and was successfully applied to the pharmacokinetic study of scopolin in rats.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylamiodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether-acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol-acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C18 column operating in combination with a radial compression separation unit and a methanol-25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay. The limit of sensitivity of the assay was 0.025 micrograms/ml with a precision of +/- 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

High performance liquid chromatographic-mass spectrometric assay for the quantitation of BMS-204352 in dog K(3)EDTA plasma

A high performance liquid chromatographic-mass spectrometric (LC/MS) assay was developed and validated for the determination of BMS-204352 in dog K(3)EDTA plasma. A 0.5 mL aliquot of control plasma was spiked with BMS-204352 and internal standard (IS) and buffered with 1 mL of 5 mM ammonium acetate. The mixture was then extracted with 3 mL of toluene. After separation and evaporation of the organic phase to dryness using nitrogen at 40 degrees C, the residue was reconstituted in the mobile phase and 25 microL of the sample were injected onto a Hypersil C(18) column (2 x 50 mm; 3 microm) at a flow rate of 0.5 mL/min. The mobile phase was consisted of two solvent mixtures (A and B). Solvent A was composed of 5 mM ammonium acetate and 0.1% triethylamine in 75:25 v/v water:methanol, pH adjusted to 5.5 with glacial acetic acid, and solvent B was 5 mM ammonium acetate in methanol. A linear gradient system was used to elute the analytes. The mass spectrometer was programmed to admit the de-protonated molecules at m/z 352.7 (IS) and m/z 357.9 (BMS-204352). Standard curves of BMS-204352 were linear (r(2) > or = 0.998) over the concentration range of 0.5-1000 ng/mL. The mean predicted quality control (QC) concentrations deviated less than 5.1% from the corresponding nominal values (ie 4, 80, 400 and 2000 ng/mL); the within- and between-assay precision of the assay were within 5.5% relative standard deviation. Stability of BMS-204352 was confirmed after at least three freeze/thaw cycles and BMS-204532 was stable in dog plasma when stored frozen at or below -20 degrees C for at least 16 weeks in spiked QC samples and for at least 4 1/2 weeks for in vivo study samples. BMS-204352 and IS were stable in the injection solvent at room temperature for at least 24 h. The assay was applied to delineate the pharmacokinetic disposition of BMS-204352 in dogs following a single intravenous dose administration. In conclusion, the assay is accurate, precise, specific, sensitive and reproducible for the pharmacokinetic analysis of BMS-204532 in dog plasma.     

Simple High-Performance Liquid Chromatographic Method for the Analysis of Quinine in Human Plasma without Extraction

Abstract

A simple, rapid and sensitive assay, capable of quantitating quinine (Q) in human plasma samples is reported. The assay uses a reversed-phase C18 HPLC column packed with 5 μ ODS Hypersil. The chromatographic separation was accomplished with an isocratic mobile phase comprising acetonitrile-aqueous phosphate buffer pH 2 (50:50, v/v) containing 25 mM sodium dodecyl sulfate and 3 mM tetrabutylammonium bromide at a flow rate of 0.5 ml/min. The eluant was monitored by a fluorescence detector (excitation wavelength at 350 nm and emission wavelength at 450 nm). The assay was based on a simple plasma protein precipitation technique. To 200 μ of plasma sample, 400 μ of internal standard (cinchocaine 30 μ/ml in methanol) was added. After brief vortexing and centrifugation, the clear supernatant was injected onto the HPLC column. The inter- and intra-assay coefficients of variation were found to be less than 10%. The lowest limit of detection for Q in plasma was 18 ng/ml.     

Simultaneous quantification of methylene blue and its major metabolite,azure B,in plasma by LC‐MS/MS and its application for a pharmacokinetic study

A simple and sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC‐UV method for quantifying etoposide in plasma and tumor interstitial fluid by microdialysis: application to pharmacokinetic studies

A simple and sensitive bioanalytical method was developed and validated for determination of etoposide in plasma and microdialysis samples of Walker‐256 tumor‐bearing rats. A microdialysis probe was implanted in the center of a subcutaneous tumor and Ringer's solution was used as perfusion medium. Chromatographic separation was conducted on a Shimadzu CLC‐C₈ column using a mobile phase consisting of water–acetonitrile (70:30; v/v) adjusted to pH 4.0 ± 0.1 with formic acid at a gradient flow rate of 1.0–0.6 mL/min, an injection volume of 30 μL and UV detection at 210 nm. Microdialysate samples were analyzed without processing and plasma samples (100 μL) were spiked with phenytoin as internal standard (IS) (1 µg/mL) followed by extraction with tert‐butyl methyl ether. The organic layer was evaporated and reconstituted with 100 μL of mobile phase before injection. The methods for plasma and microdialysate were linear in the ranges of 25–10,000 ng/mL and of 10–1500 ng/mL, respectively. All the validation parameters such as intra‐ and inter‐day precision and accuracy and stability were within the limits established by international guidelines. The present method was successfully applied in the investigation of etoposide pharmacokinetics in rat plasma and microdialysate tumor samples following a single 15 mg/kg intravenous dose. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a sensitive and rapid UPLC‐MS/MS method for the determination of koumine in rat plasma: application to a pharmacokinetic study

A rapid, selective and sensitive method using UPLC‐MS/MS was first developed and validated for quantitative analysis of koumine in rat plasma. A one‐step protein precipitation with methanol was employed as a sample preparation technique. Plasma samples were separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, i.d. 1.7 µm) with a gradient mobile phase consisting of methanol with 0.1% (v/v) formic acid and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. Detection and quantification were performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization. Good linearity (r > 0.9997) was achieved using weighted (1/x²) least squares linear regression over a concentration range of 0.025–15 µg/mL with a lower limit of quantification of 0.025 µg/mL for koumine. The intra‐ and inter‐ precisions (relative standard deviation) of the assay at all three quality control samples were 5.6–14.1% with an accuracy (relative error) of 5.0–14.0%, which meets the requirements of the US Food and Drug Administration guidance. This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous dose of 20 mg/kg koumine. Copyright © 2012 John Wiley & Sons, Ltd.     

Glycoalkaloid content in pet food by UPLC-tandem mass spectrometry

The glycoalkaloid content of pet food containing potatoes is investigated using a liquid-liquid solvent extraction followed by analysis by ultra-high pressure liquid chromatography tandem mass spectrometry (UPLC-MS-MS). Pet food samples are homogenized and extracted with a solution of 50:50 (v/v) acetonitrile-deionized water containing 5% acetic acid. Following vortexing and centrifugation, 3 mL of the supernatant is filtered and diluted in deionized water. The extract is injected onto a reverse phase C18 UPLC column with an initial mobile phase composed of 0.15% acetic acid in water (A) and 0.15% acetic acid in methanol (B) in a ratio of 70:30, respectively. The mobile phase reaches a final concentration of 15% A and 85% B over 10 min, at which point it is returned to the initial conditions. α-Solanine is measured by monitoring transitions m/z = 868.50 → 398.40 and 868.50 → 722.50, while α-chaconine is measure by monitoring transitions m/z = 852.60 → 97.80 and 852.60 → 706.50. Each analyte is measured and combined to determine total glycoalkaloid content (TGA). The results of the analysis of 52 pet food samples indicate both glycoalkaloids are present in all samples and two pet foods were found to contain > 100 μg/g total glycoalkaloid.     

Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for on-line sample clean-up

A new automated method for the quantitative analysis of cyproterone acetate (CPA) in human plasma has been developed using on-line solid phase extraction (SPE) prior to the LC-MS/MS determination. The method was based on the use of a pre-column packed with internal-surface reversed-phase material (LiChrospher RP-4 ADS, 25 mm x 2 mm) for sample clean-up coupled to LC separation on an octadecyl silica stationary phase by means of a column switching system. A 30 microl plasma sample volume was injected directly onto the pre-column using a mixture of water, acetonitrile and formic acid (90:10:0.1 (v/v/v)) adjusted to pH 4.0 with diluted ammonia as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase consisting of water, methanol and formic acid (10:90:0.1 (v/v/v)). The dispensing flow rates of the washing liquid and the LC mobile phase were 300 microl min(-1). Medroxyprogesterone acetate (MPA) was used as internal standard. The MS ionization of the analytes was achieved using electrospray (ESI) in the positive ion mode. The pseudomolecular ionic species of CPA and MPA (417.4 and 387.5) were selected to generate daughter ions at 357.4 and 327.5, respectively. Finally, the developed method was validated according to a new approach using accuracy profiles as a decision tool. Very good results with respect to accuracy, detectability, repeatability, intermediate precision and selectivity were obtained. The LOQ of cyproterone acetate was 300 pg ml(-1).     

Influence of organic solvents in the mobile phase on the determination of carboxylic acids and inorganic anions in grape juice by ion chromatography

Investigations have been initiated to develop a sufficiently good separation of both major and minor organic acids and inorganic anions present in grape musts using a Dionex As11 column, a sodium hydroxide gradient elution and a suppressed conductivity detection. Separation was complicated in aqueous mobile phase by co-elutions and selectivity was optimized using organic modifiers that alter ion-exchange selectivity for hydrophobic ions. In this study, the influence of three different solvents (methanol, ethanol and acetonitrile) on the efficiency of column was compared. The best separation of all ions in a synthetic solution was achieved with an eluent containing 13% (v/v) methanol and 13% (v/v) ethanol in water, the run during only 20 min. This method was next applied to grape juices with success and has shown sensitivity and reproducibility. Moreover, sample preparation was a simple 20-fold dilution with 0.45 microm filtration and direct injection without prior sample clean-up.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of lisinopril in human plasma

A simple and sensitive liquid chromatography/tandem mass spectrometry method employing electrospray ionization, to quantify lisinopril in human plasma using pseudoephedrine hydrochloride as the internal standard (IS), has been developed and validated. A mixture of methanol and 0.1% formic acid in water (50:50, v/v) was used as the isocratic mobile phase. A simple liquid-liquid extraction procedure was used as sample preparation method. The method validation demonstrated the specificity, lower limit of quantification, accuracy, and precision of measurements. Selected reaction monitoring was specific for lisinopril and pseudoephedrine hydrochloride; no endogenous materials from blank plasma interfered with the analysis of lisinopril or the IS. The assay was linear over the concentration range 0.78-100 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9984-0.9998. The intra- and inter-day precision, determined for quality control samples, were less than 4.18%. The method was employed in a pharmacokinetic study after oral administration of 10 mg lisinopril to 20 healthy volunteers.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

HPLC with inductively coupled plasma optical emission spectrometric detection for the analysis of inositol phosphates

The use of inductively coupled plasma optimal emission spectroscopy as a detector for the high-performance liquid chromatographic analysis of inositol phosphates is studied. It is found that separation of different inositol phosphates with a mobile phase consisting of tetraethylammonium (0.14%, w/v), methanol (5%, v/v), and formic acid (0.18%, w/v) may be obtained on a PRP-1 column with an analysis time of 18 min. In addition, high specificity and sensitivity of the detection system used permits detection of the inositol phosphates from bi- to hexaphosphate free from interference of other chromatographic peaks, which could be from the sample or mobile phase. Additionally, it is possible to use less sample because of the high sensitivity of the detection system.     

Liquid chromatographic/electrospray mass spectrometric determination (LC/ESI-MS) of chelerythrine and dihydrochelerythrine in near-critical CO2 extracts from real and spiked plasma samples

Two polar benzo[c]phenanthridine alkaloids, chelerythrine (CHE) and dihydrochelerythrine (DHCHE), were extracted at 35 °C and 10 MPa (15 MPa for real samples) from real and spiked plasma samples with acceptable recoveries (95.1% and 81.0%, respectively) using near-critical CO₂ modified with aqueous (1:1, v/v) methanol. The alkaloids were quantified by a liquid chromatographic/electrospray mass spectrometric (LC/ESI-MS) method on a Zorbax SB-CN column (75 mm × 4.6 mm, 3.5 μm particle size) using methanol (organic phase) and 50 mM ammonium formiate (aqueous phase) as a mobile phase. A linear gradient 0-1 min, isocratic at 60% organic phase (v/v); from 1.0 to 7.0 min, 60-71% organic phase (v/v); from 7.0 to 18.0 min, 71-60% organic phase (v/v) was applied. The limit of detection was 1.22 ng (3.50 pmol) for CHE and 0.95 ng (2.72 pmol) for DHCHE per 1 ml of the sample. The linearity of the calibration curves was satisfactory as indicated by coefficients of determination 0.9979 and 0.9995 for CHE and DHCHE, respectively. Repeatability and intermediate precision (average R.S.D.s) were 1.0-1.5%, accuracy was in the range 99.7-100.3%. Average recovery was 100.1% for both, standard solutions and spiked plasma extracts. Three samples of real rat plasma were extracted and analysed to test the method.     

Simultaneous quantitation of HIV-protease inhibitors ritonavir, lopinavir and indinavir in human plasma by UPLC-ESI-MS-MS

A selective, sensitive and high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of HIV-protease inhibitors ritonavir (RTV), lopinavir (LPV) and indinavir (IDV) in human plasma. Sample clean-up involved protein precipitation of both drugs and fluconazole used as internal standard from 100 μL human plasma. All the analytes were chromatographically separated on a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm particle size) analytical column using 0.1% formic acid and methanol (40:60, v/v) as the mobile phase. The parent → product ion transitions for ritonavir (m/z 721.40→ 296.10), lopinavir (m/z 629.40→ 447.40) and indinavir (m/z 614.4→ 421.0) IS (m/z 307.10 → 220.10) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was validated over the concentration range of 30-15,000 ng/mL for LPV and IDV and 3-1500 ng/mL for RTV. The method was successfully applied to a pilot bioequivalence study in 36 healthy human subjects after oral administration of lopinavir 200 mg and ritonavir 50 mg tablet formulation under fasting conditions.     

Development and validation of an LC-MS-MS method for the simultaneous determination of sulforaphane and its metabolites in rat plasma and its application in pharmacokinetic studies

A highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay is developed and validated for the quantification of sulforaphane and its metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione (SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from rat plasma by methanol-formic acid (100:0.1, v/v) and analyzed using a reversed-phase gradient elution on a Develosil 3 μm RP-Aqueous C(30) 140? column. A 15-min linear gradient with acetonitrile-water (5:95, v/v), containing 10 mM ammonium acetate and 0.2% formic acid, as mobile phase A, and acetonitrile-water (95:5, v/v), containing 10 mM ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The calibration curves are linear over the concentration range of 25-20,000 ng/mL of plasma for each analyte. This novel LC-MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in rats.     

超高效液相色谱-串联质谱法测定茶叶、代用茶和饮料食品中63种非法添加化合物

建立了超高效液相色谱-串联质谱（UPLC-MS/MS）同时测定茶叶、代用茶和饮料食品中63种非法添加化合物的分析方法。样品经甲醇超声提取后,采用Thermo Acclaim RSLC C18色谱柱（100 mm×2.1 mm,2.2 μm）分离,以5 mmol/L甲酸铵溶液（含体积分数为0.1%的甲酸）-0.1%（体积分数）甲酸乙腈为流动相进行梯度洗脱。在电喷雾离子源正离子模式下,采用动态多反应监测（dMRM）方式测定,外标法定量。63种待测化合物在相应的线性范围内呈良好的线性关系,相关系数（R²）均大于0.99;定量限为0.10~2.50mg/kg;在3个添加水平下,63种待测物的平均回收率为62.4%~129.4%,进样精密度和重复性的相对标准偏差为0.3%~9.6%（n=6）。该方法简便快捷、准确可靠,适用于茶叶、代用茶和饮料食品中非法添加具有解热镇痛效果的化合物检测。     

Determination of the N-methyl-D-aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl-D-aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75x4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate-ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250x4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.