Journal of Chromatography B (v.879, #1)

2-tert-Butylprimaquine (NP-96) is a novel quinoline anti-malarial compound with superior therapeutic profile than primaquine (PQ). Moreover, it is the first 8-aminoquinoline that is established to be devoid of methemoglobin toxicity. The purpose of the present study was to investigate covalent adduct formation tendency of PQ, NP-96 and their phase I metabolites with glutathione (GSH) and N-acetylcysteine (NAc). For the same, the two compounds were incubated in human and rat liver microsomes in the presence of trapping agents and NADPH. In a control set, NADPH was excluded, while a blank was also studied that was devoid of both NADPH and microsomes. The components in the reaction mixtures were initially separated on a C-18 column (250 mm × 4.6 mm, 5 μm) using a mobile phase composed of acetonitrile and 10 mM ammonium acetate in a gradient mode. The samples were then subjected to LC–MS n and LC–HR-MS analyses, and data were collected in full scan MS, data dependent MS/MS, targeted MS/MS, neutral loss scan (NLS) and accurate mass (MS/TOF) modes. In a significant finding, both PQ and NP-96 themselves showed potential to bind covalently with GSH and NAc, as adducts were observed even in the control and blank incubations. Intense peaks corresponding to covalent adduct of mono-hydroxy metabolite of NP-96 with GSH and NAc were also detected in NADPH supplemented reaction solution.
Keywords: Primaquine; NP-96; Glutathione; N-Acetylcysteine; Reactive metabolite; LC–MS;

Amphetamine-type stimulants (ATS) are a group of chiral amine drugs which are commonly abused for their sympathomimetic and stimulant properties. ATS are extensively metabolised by hepatic cytochrome P450 enzymes. As metabolism of ATS has been shown to be highly stereospecific, stereoselective analytical methods are essential for the quantitative determination of ATS concentrations for both in vivo and in vitro studies of ATS metabolism. This paper describes a new stereoselective method for the simultaneous determination of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), 3,4-hydroxymethamphetamine (HHMA) and 3,4-hydroxyamphetamine (HHA) in human urine samples validated according to the United States Food and Drug Administration guidelines. In this method, analytes are simultaneously extracted and derivatized with R-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-MTPCl) as the chiral derivatization reagent. Following this, the analytes were subjected to a second derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) which targets the hydroxyl groups present in HMMA, HMA, HHMA and HHA. The derivatized analytes were separated and quantified using gas chromatography–mass spectrometry (GC–MS). The method was evaluated according to the established guidelines for specificity, linearity, precision, accuracy, recovery and stability using a five-day protocol. Intra-day precision ranged from 0.89 to 11.23% RSD whereas inter-day precision was between 1.03 and 12.95% RSD. Accuracy values for the analytes ranged from −5.29% to 13.75%. Limits of quantitation were 10 μg/L for AM, MA, MDMA, HMA and HMMA and 2 μg/L for MDA, HMA and HHA. Recoveries and stability values were also within accepted values. The method was applied to authentic ATS-positive samples.
Keywords: Amphetamine-type stimulants; GC–MS; Enantiomers; Chiral derivatization; Liquid–liquid extraction;

A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was developed for the determination of dexamethasone (Dex) and dexamethasone sodium phosphate (Dex SP) in plasma and human cochlear perilymph. After proteins were precipitated with a mixture of acetonitrile and methanol, Dex, Dex SP and flumethasone, the internal standard, were resolved on a C18 column using gradient elution of 5 mM ammonium acetate and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.5–500 μg/L (r  > 0.99), bias was <±10%, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.5 μg/L for both Dex and Dex SP. The assay has been used successfully in a clinical pharmacokinetics study of Dex and Dex SP in cochlear perilymph and plasma.
Keywords: Dexamethasone; Dexamethasone sodium phosphate; Cochlear perilymph; Plasma; LC–MS/MS;

Protein phosphorylation activates or deactivates many other proteins especially protein enzymes, and plays a significant role in a wide range of cellular processes. Recent advances in phosphopeptide enrichment procedures and mass spectrometry-based peptide sequencing techniques have enabled us to identify large number of protein phosphorylation sites. In this study, we combined three different HPLC techniques in fractionating enriched phosphopeptides before RPLC–MS/MS analysis, and found that although between 4000–5000 unique phosphopeptides could be identified following any of the HPLC fraction method, different HPLC method yielded a considerable amount of non-overlapping unique phosphopeptides. Combining data from all the HPLC methods, we were able to identify 9069 unique phosphopeptides and 3260 phosphoproteins covering 9463 unique phosphorylation sites, indicating that different HPLC methods are complementary to each other, and can be used together in order to increase the phosphoproteome coverage. A number of new phosphorylation sites and novel phosphorylation motifs were also discovered from our study.
Keywords: HPLC; SCX; HILIC; ERLIC; IMAC; Phosphopeptide;

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been applied for the quantitative determination of β-blocker drugs in one-drop of human serum samples using drop-to-drop solvent microextraction (DDSME) as a preconcentrating probe. The optimum experimental conditions for β-blocker drugs were investigated and 1.8 μL volume of toluene for 10 min extraction time with the 5% addition of NaCl under pH 11.0 was found to be the best conditions for the separation and preconcentration of drugs from 30 μL of serum sample from a patient with high blood pressure. The optimized methodologies for DDSME/MALDI-MS analyses exhibited a good linearity with intra- and inter day precision value of 8.5–10.5% and 9.4–12.6%, respectively. The proposed DDSME/MALDI-MS offers a very simple, rapid and low-cost technique for the determination of β-blocker drugs in one drop of serum sample. The reported method has been successfully applied for the determination of propranolol and nadolol in small volume of serum sample from patient suffering from high blood pressure. In future, this technique could be applied for pharmacokinetic and clinical studies.
Keywords: Matrix-assisted laser desorption/ionization mass spectrometry; Drop-to-drop solvent microextraction; β-Blocker drugs; Serum;

A sensitive LC–MS/MS method for the quantitative analysis of the Echinacea purpurea constituent undeca-2-ene-8,10-diynoic acid isobutylamide in human plasma by Andrew K.L. Goey; Rolf W. Sparidans; Irma Meijerman; Hilde Rosing; Jan H.M. Schellens; Jos H. Beijnen (41-48).
Echinacea purpurea is one of the most popular herbal medicines and is known for its immunostimulatory effects. Alkylamides are the main lipophilic components of E. purpurea that contribute to its pharmacological actions. For quantification in human plasma of one of these alkylamides, undeca-2-ene-8,10-diynoic acid isobutylamide, a sensitive LC–MS/MS assay has been developed and validated. Plasma samples were pretreated using liquid–liquid extraction with a mixture of diethyl ether and n-hexane (50:50, v/v). Dried extracts were reconstituted in 50 μL of acetonitrile–water (50:50, v/v) after which 15 μL of sample was injected into the HPLC system. HPLC was performed using a Polaris 3 C18-A column (50 mm × 2 mm ID) and isocratic elution with acetonitrile–water (50:50, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. Subsequently, electrospray ionization in the positive ion mode followed by tandem mass spectrometry was performed for detection. The total run time was 3 min. The assay was validated over a concentration range from 0.05 to 50 ng/mL for undeca-2-ene-8,10-diynoic acid isobutylamide, with 0.05 ng/mL being the lower limit of quantification using 1.0 mL plasma samples. Inter-assay inaccuracy (±12.7%), within-day and between-day precisions (CV ≤ 8.23%) were acceptable. Further, undeca-2-ene-8,10-diynoic acid isobutylamide was found to be chemically stable under relevant conditions. Finally, the applicability of this assay has been successfully demonstrated in a pharmacokinetic experiment in which a human volunteer ingested a commercial extract of E. purpurea.
Keywords: Undeca-2-ene-8,10-diynoic acid isobutylamide; Echinacea purpurea; Alkylamide; Isobutylamide; LC–MS/MS; Human plasma;

Differential hydrolysis of homocysteine thiolactone by purified human serum 192Q and 192R PON1 isoenzymes by Ahmet Bayrak; Tülin Bayrak; Ediz Demirpençe; Kamer Kılınç (49-55).
Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human 192Q and 192R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for 192R PON1 and 590 for 192Q PON1. The final purified enzymes were shown as single protein bands close to 45 kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K m values of 192Q and 192R PON1 for homocysteine thiolactone were 23.5 mM and 22.6 mM respectively. For 192R PON1, the V max was 2.5-fold and k cat/K m was 2.6-fold higher than those for 192Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining 192Q and 192R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.
Keywords: Paraoxonase 1; Purification; Polymorphism; Atherosclerosis; Homocysteine thiolactone;

Acetylcholine (ACh) associated with Alzheimer's and Parkinson's disease is the major neurotransmitter in vertebrates. In support of clinical studies on the mechanism of the illnesses and development of medicines for these diseases, the LC–ESI-MS/MS method was developed and validated for the direct quantification of ACh in dialysate samples with acetylcholine-D9 bromide (IS) as the isotope-labeled internal standard. The analytes were separated on the Waters Hilics C18 Column (2.1 mm × 100 mm, 3 μm) on LC with mobile phase ultrapure water–200 mM ammonium formate (pH 3.04)–acetonitrile (30:5:65, vol/vol/vol) at a flow rate of 300 μL/min, and monitored with a fragment ion of m/z 87 formed from a molecular ion of m/z 146 for ACh and that of m/z 87 from m/z 155 for IS during multiple reaction monitoring (MRM) positive ion mode. The lower limit of quantitation (LLOQ) of ACh was lower than 0.1 nmol/L in dialysate samples, equivalent to 0.2 fmol injected on-column. The developed method could be utilized in the analysis of ACh in dialysate samples and these results were in good agreement with the gradient elution study.
Keywords: Acetylcholine; Microdialysis; LC–MS/MS;

Development and validation of a HPLC method for the quantification of baculovirus particles by Julia Transfiguracion; Jimmy A. Mena; Marc G. Aucoin; Amine A. Kamen (61-68).
A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37 °C for a minimum of 1 h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris–HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1 × 108 to 5.0 × 1010  viral particles (VP/ml). The detection limit was 3.0 × 107 and the quantification limit was 1 × 108  VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.
Keywords: Baculovirus; HPLC; Quantification; Total virus particle; Viral genome labeling;

Investigation of piceid metabolites in rat by liquid chromatography tandem mass spectrometry by Donggeng Wang; Zhiwen Zhang; Jianfeng Ju; Xinyang Wang; Wenjing Qiu (69-74).
There is considerable evidence that stilbenes provide health benefits. Trans-piceid is one of the major stilbenoid compounds in red wine and other plants. The purpose of this study is to investigate the metabolism of piceid in rats, including its conversion product by intestinal microflora in vitro and urinary metabolites. A HPLC–MS/MS method with electrospray ionization (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of piceid. Three metabolites resveratrol, dihydropiceid and dihydroresveratrol were detected after incubating with gut microbiota for 5 h. Four urinary metabolites of piceid were identified as resveratrol, dihydroresveratrol monosulfate, piceid monosulfate and piceid monoglucuronide.
Keywords: Piceid; HPLC–MS–MS; In vitro metabolism; Urinary metabolite;

Screening of thiourea derivatives and carbonyl-2-aminothiazole derivatives for potential CCR4 antagonists using capillary zone electrophoresis by Shuyu Zhang; Hui Qi; Pazilaiti Yakufu; Fang Zhao; Xiaomei Ling; Junhai Xiao; Ying Wang (75-82).
CC chemokine receptor 4 (CCR4) is a kind of G-protein-coupled receptors with a characteristic seven-transmembrane structure and selectively expressed on Th2-type CD4+ T-cells. CCR4 has been identified as a potentially important drug target for the treatment of T cell-mediated allergic inflammatory diseases. In this study, a novel series of CCR4 antagonists were screened by investigating the interactions between the compounds and the human CCR4 N-terminal peptide ML40 using capillary zone electrophoresis (CZE) for the first time. Both qualitative and quantitative characterizations of the compound-peptide binding were determined. The results showed that, compared with positive control, ten of the compounds were interacted with ML40, which were A3C223, A3C231, A4C238, A3C241, A4C241, A4C239, ZXF0337, ZXF0432, ZXF0519 and ZXF0637A, and their binding constants were calculated from the Scatchard plot by regression. The binding constants of the compounds to ML40 were calculated and the binding constant of ZXF0432 was the largest among them [(7.6334 ± 0.1907) × 104  M−1]. Here, a sensitive and selective high-performance analytical method based on CZE was developed for screening of thiourea derivatives and C-arbonyl-2-aminothiazole derivatives for potential CCR4 antagonists for the first time. The methodology presented should be generally applicable to study compounds-ML40 interactions as a powerful, sensitive and fast screening method for CCR4 antagonist discovery.
Keywords: Capillary zone electrophoresis; CCR4 antagonists/interactions; Thiourea derivatives; C-arbonyl-2-aminothiazole derivatives;

Elevation of plasma homocysteine concentration is recognized as an independent predictor of cardiovascular disease risk. Therefore, quantification of homocysteine and related sulphur amino acids cysteine and methionine from plasma samples is routinely performed in clinical laboratories. Due to the highly hydrophilic character of these amino acids, previously reported LC–MS methods often suffered from very short chromatographic retention resulting in inadequate separation from matrix background and possible co-eluents. In the present method, aqueous normal phase (ANP) chromatography was introduced to improve chromatographic separation for liquid chromatography–electrospray ionization tandem mass spectrometry. Selective qualification of analytes and internal standards was achieved by qualifier ion monitoring. Using this enhanced selectivity, spurious co-eluents were identified and separated from the analyte signal by optimization of chromatographic conditions. Method validation proved high precision and accuracy (intra-assay reproducibility 1.2–4.3% CV, inter-assay reproducibility 3.4–6.1% CV, accuracy 91.3–105.9%). Total cycle time of 7 min and low costs per sample allow high-throughput application in clinical diagnostics and research trials.
Keywords: Homocysteine; Cysteine; Methionine; LC–MS/MS; Aqueous normal phase chromatography; Qualifier ions;

A new pretreatment method, solid-phase extraction combined with dispersive liquid–liquid microextration (SPE–DLLME), was proposed in first time for the determination of clenbuterol (CLB) in porcine tissue samples. The tissue samples were firstly extracted by SPE, then its eluents were used as dispersant of the followed DLLME for further purification and enrichment of CLB. Various parameters (such as the type of SPE sorbent, the type and volume of elution solvent, the type and volume of extractant and dispersant, etc.) that affected the efficiency of the two steps were optimized. Good linearity of CLB was ranged from 0.19 μg/kg to 192 μg/kg with correlation coefficient (r 2) of 0.9995. The limit of detection (LOD) was 0.07 μg/kg (S/N = 3) and the recoveries at three spiked levels were ranged from 87.9% to 103.6% with the relative standard deviation (RSD) less than 3.9% (n  = 3). Under the optimized conditions, the enrichment factor (EF) for CLB could up to 62 folds. The presented method that combined the advantages of SPE and DLLME, had higher selectivity than SPE method and was successfully applied to the determination of CLB in tissue samples.
Keywords: Solid-phase extraction; Dispersive liquid–liquid microextraction; Clenbuterol; Tissue samples; Liquid chromatoraphy;

Meclizine is an antihistamine and has been widely used for prophylactic treatment of motion sickness. To facilitate its pharmacokinetic study in human subjects, a high performance liquid chromatography–mass spectrometric method employing positive electrospray ionization was developed for the determination of meclizine concentration in human plasma.Meclizine together with the internal standard (flunarizine) was extracted from 0.1 ml of human plasma by protein precipitation using acetonitrile. The chromatography was performed using a Zorbax SB-C18 column (150 × 2.1 mm, 5 μm, Agilent) with the mobile phase consisting of acetonitrile and 0.2% formic acid containing 2 mM amino acetate. Multiple reaction monitoring was used for quantification. The validation of the method including sensitivity, linearity, reproducibility and stability was examined.The lower limit of quantification (LLOQ) of the developed assay method for meclizine was 0.5 ng/ml and the linear calibration curve was acquired with R 2  > 0.99 between 0.5 and 200 ng/ml. The intra-day and inter-day variation of the current assay was evaluated with the coefficient of variations (CVs%) within 12.92% at LLOQ and 7.15% for other quality control samples, whereas the mean accuracy ranged from 99.2% to 102.7%. The samples were stable under the storage conditions at least for a month.The present method provides a robust, fast and sensitive analytical tool for meclizine in human plasma and has been successfully applied to a clinical pharmacokinetic study in 20 subjects.
Keywords: Meclizine; Liquid chromatography; Mass spectrometry; Pharmacokinetics;

Biochemical and binding characteristics of boar epididymal fluid proteins by Pavla Maňásková-Postlerová; Nina Davidová; Věra Jonáková (100-106).
During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12–17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12–18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm–zona pellucida binding.
Keywords: Epididymal proteins; RP HPLC; Biotin-labeled ligands;

An effective, rapid and reliable capillary electrophoresis–laser induced fluorescence (CE–LIF) procedure was built to study the characterization of tyrosine kinase (TK), which was a target for drug screening. In this procedure, CE separated the sample of the TK reaction and LIF selectively detected the fluorescence-labeled polypeptide substrate and product. The precise TK activity was quantitated by introducing the transformation ratio of the substrate (T%) to avoid the deviation resulted from the detection sensitivity and the injection amounts in different runs and different capillaries. By measuring the T%, the effects of various reaction conditions were optimized. Meanwhile, the progression of the enzyme reaction was monitored. The K m and V max were calculated for TK under the optimized experimental conditions. In addition, the inhibition effectiveness of two model inhibitors, Staurosporine and SU6656 were evaluated. The results indicated that the screening platform based on electrophoresis was suitable for TK analysis and laid a foundation for the HTS of TK inhibitors.
Keywords: Tyrosine kinase; Inhibitor screening; Capillary electrophoresis; Laser-induced fluoresce; The transformation ratio of the substrate;

This study was aimed at developing a simple HPLC method for the detection of daidzein in rat plasma. Daidzein was extracted from rat plasma with ethylparaben as internal standards (IS). Chromatographic separation of daidzein and IS was achieved by a Dikma Dimonsil C18 column (200 mm × 4.6 mm) with the mobile phase consisting of methanol–water (55:45, v/v) at a flow rate of 1.0 mL/min. The injection volume was 20 μL and the detecting wavelength was 249 nm. The calibration curve was linear over a concentration range from 0.05 to 5 μg/mL, and the accuracy was within a range of 93.4–126.2%. This HPLC method was applied successfully to the pharmacokinetic study of two kinds of daidzein-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (D-NPs) and daidzein suspension after intravenous injection in rats. Significant differences in main pharmacokinetic parameters of daidzein suspension and D-NPs were observed.
Keywords: Daidzein; HPLC; Pharmacokinetics; PLGA; Nanoparticles;

A rapid, sensitive, and environmental-friendly method has been developed for the simultaneous determination of seven neonicotinoid insecticides residues in bovine muscle and liver. The sample preparation procedure was based on a high automated pressurized solvent extraction (PSE) combined with solid-phase extraction (SPE) clean-up. The target compounds were identified and quantitatively determined by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) operated in multiple reaction monitoring mode. Average recoveries of the seven analytes from fortified samples ranged between 83.2% and 101.9%, with relative standard deviations (RSDs) lower than 10.8%. The limits of detection (LODs) and quantification (LOQs) for neonicotinoids were in the ranges of 0.8–1.5 μg kg−1 and 2.5–5.0 μg kg−1, respectively. This validated method was successively applied to the determination of neonicotinoid insecticides in real samples from markets.
Keywords: Neonicotinoids; Insecticides; Bovine muscle and liver; Pressurized solvent extraction; LC–MS/MS;

The present article describes the quantification of mirtazapine, O-desmethylvenlafaxine, quetiapine, venlafaxine, and ziprasidone (group 1), and amitriptyline, citalopram, clomipramine, clozapine, desmethylclomipramine, desipramine, imipramine, and nortriptyline (group 2) in human serum for therapeutic drug monitoring. The method was developed to replace old techniques which applied solid phase extraction and ultra-violet detection. The old methods had reached their limit of capacity regarding the number of samples and co-medicated drugs interfering with the detection. Serum samples were precipitated with zinc sulphate and methanol containing a stable isotope labelled analog for each analyte. Quantitative analysis was performed by ultra high pressure liquid chromatography combined with a tandem mass spectrometer using a Zorbax SB-C8 column (2.0 × 50 mm; 1.8 μm) with a mobile phase consisting of 0.1% formic acid in water and methanol, respectively. The total run time of the chromatography was 4 min. Precision and trueness varied from 2.6% to 14.9% and 87.6% to 103.5%, respectively. At the lower limit of quantification, precision was up to 17.9% and trueness varied from 89.5% to 111.5%. A five point standard curve covering the clinically relevant ranges with a power function fit was applied for calibration. Ion suppression from matrix effects and internal standards were thoroughly investigated and are discussed. Process efficiency rates varied from 42% to 99%. The method has shortened the response time, reduced interference from other drugs, avoided acetonitrile usage, and reduced the amount of serum needed for analysis 50-fold.
Keywords: Ultra high pressure liquid chromatography; Mass spectrometry; Antidepressants; Antipsychotics; Precipitation; Ion suppression;