Enantioselective determination of azelnidipine in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [(2)H(6)]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing alpha(1)-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583-->167 for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589-->167 for [(2)H(6)]-azelnidipine. The standard curve was linear over the studied range (0.05-20 ng/mL), with r(2)>0.997 using weighted (1/x(2)) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2-8.2% and 2.4-5.8% for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2-117.0% for (R)-(-)-azelnidipine and 100.0-107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine were 71.4-79.7% and 71.7-84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.     

Quantitative determination of ondansetron in human plasma by enantioselective liquid chromatography-tandem mass spectrometry

A sensitive and enantioselective method was developed and validated for the determination of ondansetron enantiomers in human plasma using enantioselective liquid chromatography-tandem mass spectrometry. The enantiomers of ondansetron were extracted from plasma using ethyl acetate under alkaline conditions. HPLC separation was performed on an ovomucoid column using an isocratic mobile phase of methanol-5 mM ammonium acetate-acetic acid (20:80:0.02, v/v/v) at a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 294-->170 for ondansetron enantiomers, and m/z 285-->124 for tropisetron (internal standard). The method was linear in the concentration range of 0.10-40 ng/mL for each enantiomer using 200 microL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.10 ng/mL. The intra- and inter-assay precision was 3.7-11.6% and 5.6-12.3% for R-(-)-ondansetron and S-(+)-ondansetron, respectively. The accuracy was 100.4-107.1% for R-(-)-ondansetron and 103.3-104.9% for S-(+)-ondansetron. No chiral inversion was observed during the plasma storage, preparation and analysis. The method was successfully applied to characterize the pharmacokinetic profiles of ondansetron enantiomers in healthy volunteers after an intravenous infusion of 8 mg racemic ondansetron.     

Chiral analysis of ammuxetine enantiomers in dog plasma using online SPE/liquid chromatography with tandem mass spectrometric detection after precolumn chiral derivatization

Ammuxetine (AMT), a novel chiral antidepressant candidate compound, exhibits better antidepression effects than duloxetine in different animal models. In this article, a chiral derivatization method, combined with online solid phase extraction (online SPE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), was developed for the chiral separation of AMT enantiomers after administration of racemic AMT to dogs. The derivatization reaction employed 2,3,4,6‐tetra‐O‐acetyl‐b‐glucopyr‐anosyl isothiocyanate (GITC) as a precolumn chiral derivatization reagent. A SPE column Retain PEP Javelin (10 × 2.1 mm) was used to remove proteins and other impurities in plasma samples. The enantiomeric derivatives were separated on a ZORBAX SB‐C₁₈ column (50 × 2.1 mm × 3.5 μm) with an isocratic elution procedure. The selected multiple reaction monitoring mode of the positive ion was performed and the parent to the product transitions m/z 681.0/543.1 and m/z 687.4/543.1 were used to measure the derivatives of AMT and duloxetine (internal standard) with electrospray ionization. The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The method was applied to a pharmacokinetics study of AMT racemate in dogs. The results suggested that the pharmacokinetic of AMT enantiomers might be stereoselective in dogs.     

Quantitative determination of the macrolide antibiotic tulathromycin in plasma and broncho-alveolar cells of foals using tandem mass spectrometry

The long-acting antibiotic tulathromycin is on the marked for treatment of pulmonary infection of cattle, swine and horses. To measure disposition and distribution of tulathromycin in foals, a high throughput method was developed for horse plasma (calibration range: 0.006-0.8 microg/mL) and broncho-alveolar cells (calibration range: 0.1-4.0 microg/10(9)cells) using tandem mass spectrometry. Tulathromycin was extracted from plasma and broncho-alveolar fluid using cation exchange cartridges with acetonitrile/ammonia (95:5, v/v). The chromatography was performed isocratically with a mobile phase consisting of 5 mM ammonium formiate buffer/acetonitrile (30:70, v/v). The mass spectrometer operated in selected ion mode with atmospheric pressure chemical ionization to monitor the respective MH+ ions m/z 577.3 for tulathromycin and m/z 679.3 for the internal standard roxithromycin. All quality parameters fulfilled the international criteria for bioanalytical method validation and were successfully applied to the determination of tulathromycin in plasma of foals and broncho-alveolar cells. In foals, tulathromycin after intramuscular administration was rapidly absorbed, widely distributed and slowly eliminated. It cumulated manifold in broncho-alveolar cells.     

Quantitative determination of nystatin in human plasma using LC-MS after inhalative administration in healthy subjects

The antifungal polyene antibiotics nystatin was tested in a clinical trial to describe pharmacokinetics and safety after repeated administration of Nystatin "Lederle" sterile powder in healthy volunteers. To monitor the nystatin concentration-time profile in plasma we developed a sensitive method in the range of 1-100ng/ml based on liquid chromatography coupled with tandem mass spectrometry. The target substance was separated from the biological matrix on C(18) solid-phase extraction cartridges with methanol. The Chromatography was performed isocratically using a reversed phase Caltrex Resorcinearene column. The mobile phase consisted of 5mM ammonium formate buffer and acetonitrile (40:60, v/v). The mass spectrometer works with electrospray ionization in its positive selected ion monitoring (SIM) mode using the respective MH(+) ions, m/z 926.6 for nystatin and m/z 924.4 for amphotericin B as internal standard. The method validation was performed according to the demands and international criteria for validation of bioanalytical methods and was successfully applied to the quantification of nystatin in human plasma in the pharmacokinetic trial.     

Stereoselective analysis of tiopronin enantiomers in rat plasma using high-performance liquid chromatography-electrospray ionization mass spectrometry after chiral derivatization

A specific and relatively sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) was developed for the quantitative analysis of tiopronin enantiomers in rat plasma. The method is based on the derivatization of (+)-tiopronin and (-)-tiopronin with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate (GITC) in acetonitrile. The separation of resulting diastereomic derivatives was performed on C18 column (150 mm x 2.0 mm ID, packed with 5.0 mum C(18) silica RP particle), using a mobile phase of methanol/water (containing 5.3 mM formic acid) with gradient elution. LC-MS was performed in the selected ion monitoring and positive ion mode using target ions at m/z: 575 for the diastereomic derivatives of tiopronin and m/z: 603 for the derivative of N-isobutyryl-D-cysteine (internal standard). The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The calibration curves were linear over the concentration range of 0.025-5 microg/ml for both enantiomers of tiopronin. For both enantiomers of tiopronin, the interbatch and intrabatch variability values were less than 15%, and the accuracy was within +/-17% in terms of relative error. The method was successfully applied to a pharmacokinetic study of rac-tiopronin in rat.     

Development and validation of a stereoselective liquid chromatography-tandem mass spectrometry assay for quantification of S- and R-metoprolol in human plasma

A stereoselective liquid chromatography-tandem mass spectrometry assay was developed and validated for quantification of S- and R-metoprolol at concentrations of 0.5-50 microg/L in human plasma. Metoprolol was extracted from plasma by liquid-liquid extraction with ethyl acetate (82% recovery). Chromatographic separation of the enantiomers was achieved on a chiral Chirobiotic T column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). An ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268-->191 for metoprolol. Standard curves for S- and R-metoprolol fitted quadratic functions (r(2)>or=0.9995) over the range 0.5-50 microg/L in plasma, with 0.5 microg/L representing the limit of quantification. In this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. The accuracy was within the range of 92-105%.     

The determination of terbutaline in human plasma by selected ion monitoring of the t-butyldimethylsilyl ether

A method is described for the quantitative determination of terbutaline in 2 ml human plasma. The drug is extracted from plasma as the terbutaline tetraphenylboron ion pair and determined by gas chromatography mass spectrometry of its t-butyldimethylsily ether. Salbutamol is used as internal standard. Quantification is achieved by selected ion monitoring of the ion m/z 482 derived from t-butyldimethylsilyl terbutaline and m/z 495 from t-butyldimethylsilyl salbutamol. The detection limit was estimated to be 250 pg terbutaline ml-1 plasma. The coefficient of variation at the level of 1 ng terbutaline ml-1 was 4.1% (n = 5).     

High‐Throughput Chiral LC–MS/MS Method Using Overlapping Injection Mode for the Determination of Pantoprazole Enantiomers in Human Plasma with Application to Pharmacokinetic Study

A sensitive and high‐throughput chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of R‐pantoprazole and S‐pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid–liquid extraction in 96‐well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ‐RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 μL/min. The enantiomers were quantified on a triple‐quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole‐d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r² > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R‐pantoprazole and from 92.5% to 96.5% for S‐pantoprazole and the IS‐normalized matrix factor was 0.98 to 1.07 for R‐pantoprazole and S‐pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569–575, 2016. © 2016 Wiley Periodicals, Inc.     

Determination of the enantiomers of ketamine and norketamine in human plasma by enantioselective liquid chromatography-mass spectrometry

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection has been developed and validated for the simultaneous determination of plasma concentrations of (R)- and (S)-ketamine, and (R)- and (S)-norketamine. The compounds were extracted from human plasma using solid-phase extraction and then directly injected into the LC-MS system for detection and quantification. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase based upon immobilized alpha(1)-acid glycoprotein (the Chiral AGP column). The separations were achieved using a mobile phase composed of 2-propanol-ammonium acetate buffer (10 mM, pH 7.6) (6:94, v/v), a flow-rate of 0.5 ml/min and a temperature of 25 degrees C. Under these conditions, the analysis time was 20 min. Detection of the ketamine, norketamine and bromoketamine (internal standard) enantiomers was achieved using selected ion monitoring at m/z 238.1, 224.1 and 284.0, respectively. Extracted calibration curves were linear from 1 to 125 ng/ml per enantiomer for each analyte with correlation coefficients better than 0.9993 and intra- and inter-day RSDs of less than 8.0%. The method was applied to samples from a clinical study of ketamine in pain management.     

Enantioselective determination of alprenolol in human plasma by liquid chromatography with tandem mass spectrometry using cellobiohydrolase chiral stationary phases

A fast, sensitive, and enantioselective LC-MS/MS bioanalytical method was developed and validated for the direct determination of individual alprenolol enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP) along with supported liquid extraction (SLE) procedures. Complete baseline separation of enantiomeric alprenolol was achieved within 2 min in reversed phase chromatography at 0.9 ml/min. SLE in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.500-500 ng/ml for each alprenolol enantiomer using 0.0500 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed < or = 7.3% relative standard deviation (RSD) and -6.2 to 8.0% relative error (RE) for both alprenolol enantiomers.     

Simultaneous quantification of the enantiomers of verapamil and its N-demethylated metabolite in human plasma using liquid chromatography-tandem mass spectrometry

A stereoselective bioanalytical method for the simultaneous quantification of the enantiomers of verapamil and its active main metabolite norverapamil in human plasma has been developed and validated. The samples were analysed by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) in the Selected Reaction Monitoring (SRM) mode using a deuterated internal standard. The stationary phase used for the chiral separation was a Chiral-AGP. The enantiomers of verapamil were selectively detected from those of norverapamil by the mass spectrometer due to different molecular masses, although there was a chromatographic co-elution. Thus, time-consuming procedures like achiral preseparation or chemical derivatisation could be avoided. Higher detection sensitivity than earlier published methods based on fluorescence detection was obtained, although a mobile phase of high water-content and high flow-rate was introduced into the electrospray interface (85% aqueous ammonium acetate pH 7.4 +15% acetonitrile at 0.6 ml/min). The enantiomers of verapamil and norverapamil could be quantified at levels down to 50 pg and 60 pg/500 microl plasma sample, respectively, with R.S.D. in the range of 3.6-7.8%. The presented method was successfully applied to an in vivo intestinal absorption and bioavailability study in humans, using the Loc-I-Gut method.     

A validated enantioselective assay for the simultaneous quantitation of (R)-, (S)-fluoxetine and (R)-, (S)-norfluoxetine in ovine plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS)

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantitation of (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine in ovine plasma. The analytes were extracted from ovine plasma at a basic pH using a single-step liquid-liquid extraction with methyl-tert-butyl ether. Chromatographic separation of all enantiomers was achieved using an AGP-chiral column with a run time of 10 min. (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine were quantitated at the total ion current (TIC) of multiple reaction monitoring (MRM) transitions of m/z 310.2→44.1, m/z 310.2→147.7 for (R)-, (S)-fluoxetine, and m/z 296.2→30.3, m/z 296.2→133.9 for (R)-, (S)-norfluoxetine. This method was validated for accuracy, precision, linearity, range, limit of quantitation (LOQ), selectivity, recovery, dilution integrity, matrix effect, and evaluation of carry-over. Observed accuracy ranges were as follows: (R)-fluoxetine -8.82 to 3.75%; (S)-fluoxetine -10.8 to 1.46%; (R)-norfluoxetine -7.50 to 0.37% and (S)-norfluoxetine -8.77% to -1.33%. Observed precision ranges were as follows: (R)-fluoxetine 5.29-11.5%; (S)-fluoxetine 3.91-11.1%; (R)-norfluoxetine 4.32-7.67% and (S)-norfluoxetine -8.77% to -1.33%. The calibration curves were weighted (1/X(2), n=4) and observed to be linear for all analytes with the following r(2) values: (R)-fluoxetine ≥ 0.997; (S)-fluoxetine ≥ 0.996; (R)-norfluoxetine ≥ 0.989 and (S)-norfluoxetine ≥ 0.994. The analytical range of the method was 1-500 ng/ml with an LOQ of 1 ng/ml for all analytes, using a sample volume of 300 μL.     

Analysis of flumequine enantiomers in rat plasma by UFLC‐ESI‐MS/MS

In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra‐fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid–liquid extraction using methyl tert‐butyl ether as the extraction solvent. Direct resolution of the R‐ and S‐isomers was performed on a CHIRALCEL OJ‐RH column (4.6 × 150 mm, 5 μm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5–500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43–8.68 and 94.24–106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies.     

A sensitive gas chromatographic/mass spectrometric method for the resolution and quantification of ethosuximide enantiomers in biological fluids

A modified specific, sensitive and reproducible chiral gas chromatographic (GC) method for the resolution and quantification of ethosuximide enantiomers in urine and plasma was developed. The samples were extracted by liquid-liquid extraction, using diethylether and the enantiomers were separated and quantified on a chiral gas chromatographic column (25QC2 / CYDEX- beta 0.25). The method involved the use of GC/MS instrumentation for the acquisition of data in the electron impact selective-ion monitoring mode, collecting ions characteristic of both ethosuximide and alpha, alpha - dimethyl - beta - methylsuccinimide, the internal standard and of mass-to-charge ratio (m/z) exactly equal to 55 and 70 units. The limit of quantitation of the method was 2.5 microg/ml for both urine and plasma with both enantiomers. The method proved to be linear, precise and reproducible in the 5-300 microg/ml concentration range for urine samples and in the 10-250 microg/ml concentration range for plasma samples. Future research work envisaged the application of this method in pharmacokinetic and pharmacodynamic studies.     

Bioanalytical chiral chromatographic technique and docking studies for enantioselective separation of meclizine hydrochloride: Application to pharmacokinetic study in rabbits

Enantiomeric resolution and molecular docking studies of meclizine hydrochloride on polysaccharide-based chiral stationary phase comprising cellulose tris(4-methylbenzoate) chiral selector (150 × 4.6 mm, 3.0 μm) were presented. The mobile phase used was acetonitrile:10mM ammonium bicarbonate (95:05, v/v). The developed technique was used to perform the enantioselective assay of meclizine hydrochloride in its marketed formulation. The elution order of meclizine hydrochloride enantiomers was determined by docking studies. Target compound was extracted from rabbit plasma using protein precipitation technique, followed by development of bioanalytical chiral separation method using the same matrix. Application of the method to determine pharmacokinetic parameters of meclizine hydrochloride enantiomers was performed using Phoenix WinNonlin 8.1 software. The results demonstrated stereoselective disposition of meclizine hydrochloride enantiomers in rabbits.     

Evaluation of Tetrahydropalmatine Enantiomers on the Activity of Five Cytochrome P450 Isozymes in Rats Using a Liquid Chromatography / Mass Spectrometric Method and a Cocktail Approach

The aim was to evaluate the effects of tetrahydropalmatine (THP) enantiomers on the activity of five cytochrome P450 (CYP450) isozymes in vivo. A liquid chromatography / mass spectrometric (LC‐MS) method was developed for simultaneous determination of five specific probe substrates including metoprolol (2D6), caffeine (1A2), dapsone (3A4), chlorzoxazone (2E1), and tolbutamide (2C9) in rat plasma. Analytes were separated with the mobile phase consisting of 0.1% acetic acid aqueous solution and acetonitrile in a gradient elution. The mass spectrometric detection via selected ion monitoring (SIM) was operated in both positive ion mode (for metoprolol m/z 268, caffeine m/z 195, and dapsone m/z 249) and negative ion mode (for chlorzoxazone m/z 168 and tolbutamide m/z 269) in the same run. Linear correlation was obtained (r² > 0.99) over the concentration range of 0.050–25.0 µg/mL for caffeine and dapsone, 0.025–10.0 µg/mL for metoprolol, 0.050–50.0 µg/mL for chlorzoxazone, and 0.25–100.0 µg/mL for tolbutamide. Intra‐ and interday precision were less than 12.09%. The matrix effect ranged from 87.50% to 109.25% and the absolute recoveries were greater than 70%. The method was successfully applied to evaluate the effect of THP enantiomers on the activity of CYP450 isozymes by a cocktail approach. The pharmacokinetic results of five probe drugs indicated that there were stereoselective differences between the two THP enantiomers, i.e., d‐THP had the potential to inhibit the activities of CYP2D6 and CYP1A2 isozymes, while l‐THP inhibited CYP1A2 isozyme and induced CYP3A4 and CYP2C9 isozymes. Chirality 27:551–556, 2015. © 2015 Wiley Periodicals, Inc.     

Quantitative analysis of albuterol in human plasma by combined gas chromatography chemical ionization mass spectrometry

A highly sensitive and specific quantitative assay for the determination of albuterol in human plasma, based on selected ion monitoring gas chromatography chemical ionization mass spectrometry, has been developed. The [MH]+ ions from the tri-TMS derivatives of albuterol (m/z 456) and the internal standard (2H3)albuterol (m/z 459), were assayed simultaneously by selected ion monitoring. The lower limit of quantitation is 0.25 ng ml-1 and the average assay precision (CV) for albuterol concentrations ranging from 0.25 ng ml-1 to 25 ng ml-1 is approximately 4%. This method is currently being employed for the routine quantitation of albuterol in plasma following the administration of doses therapeutically effective to man.     

Validated LC-MS/MS methods for the determination of risperidone and the enantiomers of 9-hydroxyrisperidone in human plasma and urine

Two liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods are described, one for the quantitative determination of risperidone and the enantiomers of its active metabolite 9-hydroxyrisperidone (paliperidone) in human plasma and the other for the determination of the enantiomers of 9-hydroxyrisperidone in human urine. The plasma method is based on solid-phase extraction of 200 microl of sample on a mixed-mode sorbent, followed by separation on a cellulose-based LC column with a 13.5-min mobile phase gradient of hexane, isopropanol and ethanol. After post-column addition of 10 mM ammonium acetate in ethanol/water, detection takes place by ion-spray tandem mass spectrometry in the positive ion mode. Method validation results show that the method is sufficiently selective towards the enantiomers of 7-hydroxyrisperidone and capable of quantifying the analytes with good precision and accuracy in the concentration range of 0.2-100 ng/ml. An accelerated (run time of 4.3 min) and equally valid method for the enantiomers of 9-hydroxyrisperidone alone in plasma is obtained by increasing the mobile phase flow-rate from 1.0 to 2.0 ml/min and slightly adapting the gradient conditions. The urine method is based on the same solid-phase extraction and chromatographic approach as the accelerated plasma method. Using 100 microl of sample, (+)- and (-)-9-hydroxyrisperidone can be quantified in the concentration range 1-2000 ng/ml. The accelerated method for plasma and the method for urine can be used only when paliperidone is administered instead of risperidone, as there is insufficient separation of the 9-hydroxy enantiomers from the 7-hydroxy enantiomers, the latter ones being present only after risperidone administration.     

Monitoring phospholipids for assessment of matrix effects in a liquid chromatography-tandem mass spectrometry method for hydrocodone and pseudoephedrine in human plasma

Matrix effects resulting in ion suppression or enhancement have been shown to be a source of variability and inaccuracy in bioanalytical mass spectrometry. Glycerophosphocholines may cause significant matrix ionization effects during quantitative LC/MS/MS analysis and are known to fragment to form characteristic ions (m/z 184) in electrospray mass spectrometry. This ion was used to monitor ion suppression effects in the determination of hydrocodone and pseudoephedrine in human plasma as a means to track and avoid these effects. The m/z 184 ion fragment was detected in both plasma extracts and solutions of phosphatidylcholine. Post-column infusion studies showed that the ion suppression for both drugs and internal standards correlated with the elution of phospholipids. HPLC conditions were adjusted to chromatographically resolve the peaks of interest from the phospholipids. Upon repeated injection, the elution time of the phospholipids decreased while elution of the analyte peaks remained unchanged. This resulted in co-elution and significantly affected peak shape and internal standard response for the analytes. It was decided to use the phospholipid fragment to monitor this matrix effect in validation samples. The resulting method demonstrated intra-day and inter-day precision within 4.5 and 5.6% for hydrocodone and pseudoephedrine, respectively, and accuracy within 8.9 and 8.7% for hydrocodone, and pseudoephedrine, respectively. There was no statistically significant difference in the internal standard response for the determination with and without monitoring the phospholipid fragment ion. We found that monitoring the phospholipid fragment was useful in method development to avoid the matrix effects, and in routine analysis to provide a practical way to ensure the avoidance of matrix effects in each individual sample.