Journal of Chromatography B (v.847, #2)

Quantitative determination of helicid in rat plasma by liquid chromatography–electrospray ionization mass spectrometry and its application to preliminary pharmacokinetic studies by Yuanwei Jia; Guangji Wang; Haitang Xie; Xiaochang Dai; Yanhua Wang; Wei Wang; Meijuan Xu; Rui Wang; Chen Yao (72-77).
A sensitive and selective liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) method was developed for the identification and quantification of helicid in rat plasma. The method was based on simple liquid–liquid extraction (LLE). A Kromasil C18 column (150 mm × 2.00 mm, 3.5 μm) was used as the analytical column, while a mixture of acetonitrile and 500 μM ammonium chloride was used as the mobile phase. MS detection was performed using a single quadrupole mass spectrometer in a negative selected ion monitoring (SIM) mode. The deprotonated molecules [M + Cl] at m/z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard, I.S.), respectively. The lower limit of quantification of helicid was 1 ng/ml. The method was linear in the concentration range of 1–1000 ng/ml. The intra-day and inter-day precisions (R.S.D.%) were within 10.0% for the analyte. Helicid proved to be stable during all sample storage, preparation and analytical periods. The method was successfully applied to a pharmacokinetic study in rats after intragastric administration of helicid with a dose of 50 mg/kg. Only 50 μl of rat plasma at each sampling time was needed for analysis. The proposed method enables unambiguous identification and quantification for the preliminary pharmacokinetic studies of helicid.
Keywords: Helicid; LC–ESI-MS; Pharmacokinetics;

We have developed a simple, rapid and highly sensitive method for determining a plasma or cerebrospinal fluid (CSF) concentrations of individual teicoplanin components using reversed-phase high-performance liquid chromatography followed by electrochemical detection. A linear relationship was observed between concentrations and peak heights for the teicoplanin concentration range of 0.025–10 μg/mL. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection for the major component of teicoplanin was 1.0 ng/mL (signal/noise ratio >3). Daily fluctuations of standard curves (n  = 5) were small, with coefficients of variation of 3.3%. The intra-assay precision was 5.9% (n  = 5). Inter-assay precision ranged from 2.6 to 6.8%. The method described here is suitable for clinical monitoring of teicoplanin levels in plasma or CSF level and for use in studies involving pharmacokinetics of individual teicoplanin component.
Keywords: Teicoplanin; Cerebrospinal fluid; HPLC; Electrochemical detection;

A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the simultaneous determination of yield and conversion ratio of 4-nitro-1,8-naphthalic anhydride to 4-amino-1,8-naphthalic anhydride following incubation with a crude bakers’ yeast homogenate. The analytes were separated on a C18 column in gradient mode. The detection limit of 4-amino-1,8-naphthalic anhydride is 10 ng/μl when using a 10 μl sample injection volume. The nitroreductase activity in the homogenate system can be assessed during the bioconversion process. The method can be used for the simultaneous detection of 4-hydroxylamino-1,8-naphthalic anhydride, an intermediate with limited stability.
Keywords: Nitroaryl; Nitroarene; Arylhydroxylamine; Aminoaryl; Bakers’ yeast; Nitroarylreductase; HPLC; TLC;

This method simultaneously determines epinephrine, norepinephrine, dopamine and 5-hydroxytryptamine by HPLC coupled to atmospheric pressure chemical ionization mass spectrometry, using bovine chromaffin cells to test xenobiotic neurotoxicity and the secretion alterations of these neurotransmitters as endpoint. Chromatographic separation was developed by injecting the sample without previous treatment into a reversed-phase column. The signal was recorded in selected ion mode. The lowest limit of detection was found for hydroxytryptamine, while the highest limit was for norepinephrine. The feasibility of the proposed method was checked by performing measurements of neurotransmitters during the assessment the effect of mipafox on the basal and potassium-induced secretions of chromaffin cell cultures.
Keywords: Chromaffin cell secretion; Neurotransmitters determination; HPLC–MS; Mipafox; Catecholamine;

Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes by Laura Mercolini; Roberto Mandrioli; Matteo Conti; Claudio Leonardi; Gilberto Gerra; Maria Augusta Raggi (95-102).
Methadone and buprenorphine are two of the drugs most frequently used for abstinence from illicit opioids and in the treatment of pain. A sensitive and selective high-performance liquid chromatographic method with diode array detection for the simultaneous determination of methadone, buprenorphine and norbuprenorphine has been developed. Separation of the three analytes was obtained by using a reversed-phase column (C8, 250 mm × 4.6 mm i.d., 5 μm) and a mobile phase composed of 40% phosphate buffer containing triethylamine, 50% methanol and 10% acetonitrile (final apparent pH 6.0). Loxapine was used as the internal standard. An accurate pre-treatment procedure of biological samples was developed, using solid-phase extraction with C8 cartridges (100 mg, 1 mL) and needing small amounts of plasma or urine (300 μL). The calibration curves were linear over a working range of 10.0–1500.0 ng/mL for methadone and of 5.0–500.0 ng/mL for buprenorphine and norbuprenorphine in both matrices. The limit of quantitation (LOQ) and the limit of detection (LOD) were 1.0 and 0.4 ng/mL for methadone and 0.5 and 0.2 ng/mL for both buprenorphine and norbuprenorphine, respectively. The method was successfully applied to the analysis of plasma and urine samples from patients undergoing treatment with these drugs. Precision and accuracy results were satisfactory and no interference from endogenous or exogenous compounds was found. The method is suitable for the simultaneous determination of methadone and buprenorphine in human plasma and urine for therapeutic drug monitoring purposes.
Keywords: Methadone; Buprenorphine; Norbuprenorphine; High-performance liquid chromatography; DAD detection; Biological fluids; Solid-phase extraction;

Multi-dimensional liquid chromatography is often presented as an alternative to two-dimensional (2-D) gel electrophoresis for separating complex protein mixtures. The vast majority of analytical-scale 2-D LC systems have employed either off-line fractionation or stepped gradients in the first dimension separation. The latter severely restrict flexibility in setting up the first dimension gradient. We propose a novel two-dimensional LC system that employs online fractionation of proteins into a series of small reversed phase trapping columns. These traps effectively decouple the two separation dimensions and avoid problems associated with off-line fraction collection. Flexibility in determining the gradient programs for the two separations is thus enhanced. The reduced diameter of the trapping columns concentrates analyte between chromatographic dimensions. The apparatus is coupled with online electrospray time-of-flight mass spectrometry to characterize ribosomal proteins of Caulobacter crescentus.
Keywords: Multidimensonal chromatography; 2-D LC; Proteomics; Sample preparation;

Di-iso-nonylphthalate (DINP) is the major plasticizer for polyvinylchloride (PVC) polymers. Two DINP products are currently produced: DINP 1 and DINP 2. We analyzed the isononyl alcohol mixtures (INA) used for the synthesis of these two DINP plasticizer products and thus identified 4-methyloctanol-1 as one of the major constituents of the alkyl side chains of DINP 1 (8.7%) and DINP 2 (20.7%). Based on this isomer, we postulated the major DINP metabolites renally excreted by humans: mono-(4-methyl-7-hydroxy-octyl)phthalate (7OH-MMeOP), mono-(4-methyl-7-oxo-octyl)phthalate (7oxo-MMeOP) and mono-(4-methyl-7-carboxy-heptyl)phthalate (7carboxy-MMeHP). We present a fast and reliable on-line clean-up HPLC method for the simultaneous determination of these three DINP metabolites in human urine. We used ESI-tandem mass spectrometry for detection and isotope dilution for quantification (limit of quantification 0.5 μg/l). Via these three oxidised DINP isomer standards, we quantified the excretion of all oxidised DINP isomers with hydroxy (OH-MINP), oxo (oxo-MINP) and carboxy (carboxy-MINP) functional groups. With this approach, we can for the first time reliably quantify the internal burden of the general population to DINP. Mean urinary metabolite concentrations in random samples from the general German population (n  = 25) were 14.9 μg/l OH-MINP, 8.9 μg/l oxo-MINP and 16.4 μg/l carboxy-MINP. Metabolites strongly correlated with each other over all samples analyzed (R  > 0.99, p  < 0.0001).
Keywords: DINP; Phthalate; Urine; Plasticizer; Metabolites; Biological monitoring;

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20 mM pH 9.0 borate buffer containing 30 mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DİF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50 μm internal diameter, 44 cm total length, 35.5 cm effective length) at 25 °C with the application of 3 s of hydrodynamic injection at 50 mbar pressure and a potential of 29 kV. Detection wavelength was 200 nm. Under these conditions, the migration times were found to be 5.174 min for PAR, 5.513 min for CAF, 7.195 min for DİF, and 9.366 min for PRO. Linearity ranges for the method were determined as 2–200 μg mL−1 for PAR and CAF and 3–200 μg mL−1 for PRO. Limit of detections were found as 0.6 μg mL−1 for PAR and CAF and 0.8 μg mL−1 for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.
Keywords: Micellar electrokinetic capillary chromatography; Ternary mixture; Validation;

The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel “denaturing” step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC–MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC–MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9 ng/mL and 53920.0 ng/mL with a coefficient of determination (r 2) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations.
Keywords: Protein quantitation; LC–MS/MS; Antibody binding; Kringle 5; Bioanalytical assay validation; Solid phase extraction;

The determination of gemcitabine and 2′-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry by R. Honeywell; A.C. Laan; C.J. van Groeningen; E. Strocchi; R. Ruiter; G. Giaccone; G.J. Peters (142-152).
A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC–MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24 h infusion. The method was developed using 15N3 CdR as an internal standard across the concentration range of 1–500 ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC–MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1 mm × 200 mm, 5 μm) with a 50 mM formic acid: acetonitrile (9:1) mobile phase eluted at 1 ml/min. Extracted samples were observed to be stable for a minimum of 48 h after extraction when kept at 4 °C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and 15N3 CdR (231 m/z; 115 m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, ±7.9%, 19 nM; CdR: 100.0%, ±5.3%, 22 nM, linear range LOQ to 2 μM. During 24 h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no “crosstalk” between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.
Keywords: Gemcitabine; Deoxycytidine; Difluorodeoxycytidine; Mass spectrometry; HPLC;

Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5 mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50 cm neutrally coated capillary at 60 °C together with a 7 M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions ≥1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.
Keywords: CpG; Oligonucleotide; Capillary gel electrophoresis; GC-rich; Secondary-structure;

Immunoaffinity chromatography of abscisic acid combined with electrospray liquid chromatography–mass spectrometry by Veronika Hradecká; Ondřej Novák; Libor Havlíček; Miroslav Strnad (162-173).
Polyclonal antibodies with high specificity for C1-immobilised (+)-cis,trans-abscisic acid (ABA) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of an immunoaffinity chromatography (IAC) gel. The detection limit of the ELISA was approximately 4.6 × 10−10  mol/L. Sensitive electrospray liquid chromatography–mass spectrometry (LC–ESI-MS) methods were also developed with detection limits below 0.1 × 10−12  mol. The IAC allowed quick, single-step processing of samples prior to the analyses. The LC–ESI-MS and LC–ELISA techniques were used for comparative estimation of endogenous ABA levels in immunoaffinity purified extracts of normal and water-stressed Nicotiana tabacum L. leaves. The analytical approaches were validated using deuterium- and tritium-labelled internal standards, respectively. The IAC method was found to be highly effective, sensitive and convenient for isolating the target analyte from plant material.
Keywords: Abscisic acid; Immunoaffinity chromatography; Liquid chromatography–mass spectrometry;

Simultaneous determination of ten antiarrhythic drugs and a metabolite in human plasma by liquid chromatography—tandem mass spectrometry by Shuijun Li; Gangyi Liu; Jingying Jia; Yun Liu; Cheng Pan; Chen Yu; Yongbao Cai; Jianying Ren (174-181).
A simple, accurate and selective LC–MS/MS method was developed and validated for simultaneous quantification of ten antiarrhythic drugs (diltiazem, amiodarone, mexiletine, propranolol, sotalol, verapamil, bisoprolol, metoprolol, atenolol, carvedilol) and a metabolite (norverapamil) in human plasma. Plasma samples were simply pretreated with acetonitrile for deproteinization. Chromatographic separation was performed on a Capcell C18 column (50 mm × 2.0 mm, 5 μm) using a gradient mixture of acetonitrile and water (both containing 0.02% formic acid) as a mobile phase at flow rate of 0.3 ml/min. The analytes were protonated in the positive electrospray ionization (ESI) interface and detected in multiple reaction monitoring (MRM) mode. Calibration curves were linear over wide ranges from sub- to over-therapeutic concentration in plasma for all analytes. Intra- and inter-batch precision of analysis was <12.0%, accuracy ranged from 90% to 110%, average recovery from 85.0% to 99.7%. The validated method was successfully applied to therapeutic drug monitoring (TDM) of antiarrhythic drugs in routine clinical practice.
Keywords: Antiarrhythic drugs; LC–MS/MS; Therapeutic drug monitoring;

Novel generic UPLC/MS/MS method for high throughput analysis applied to permeability assessment in early Drug Discovery by Jurgen Mensch; Mark Noppe; Jef Adriaensen; Anouche Melis; Claire Mackie; Patrick Augustijns; Marcus E Brewster (182-187).
A novel generic ultra performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method for the high throughput quantification of samples generated during permeability assessment (PAMPA) has been developed and validated. The novel UPLC/MS/MS methodology consists of two stages. Firstly, running a 1.5 min isocratic method, compound-specific multiple reaction monitoring (MRM) methods were automatically prepared. In a second stage, samples were analyzed by a 1.5 min generic gradient UPLC method on a BEH C18 column (50 mm × 2.1 mm). Compounds were detected with a Waters Micromass Quattro Premier mass spectrometer operating in positive electrospray ionization using the compound-specific MRM methods. The linearity for the validation compounds (caffeine, propranolol, ampicillin, atenolol, griseofulvin and carbamazepine) typically ranges from 3.05 nM to 12,500 nM and the limits of detection for all generically developed methods are in the range between 0.61 nM and 12 nM in an aqueous buffer. The novel generic methodology was successfully introduced within early Drug Discovery and resulted in a four-fold increase of throughput as well as a significant increase in sensitivity compared to other in-house generic LC/MS methods.
Keywords: UPLC; UPLC/MS/MS; MRM; Quanoptimize; PAMPA; Permeability; High throughput;

To evaluate if pulmonary delivery of microparticles loaded with a prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), can target alveolar macrophages (AM) and reduce metabolism of INH, an HPLC–MS/MS assay with automated online extraction for quantification of INH and its metabolite acetylisoniazid (AcINH) in plasma and AMs was developed and validated. Reproducibility in rat plasma and homogenate of a rat AM cell line, NR8383, for INH and AcINH showed excellent precision and accuracy with calibration curves exhibiting linearity within a range of 1–250 ng/ml of INH and 0.05–50 ng/ml of AcINH (r 2  > 0.99). The validated methods were successfully applied to pharmacokinetic study of INHMS-loaded microparticles in rats, demonstrating efficient targeting of AMs and reduction of INH metabolism.
Keywords: Acetylated isoniazid; Isoniazid; LC/LC–MS/MS; Microparticles; PLA; Pharmacokinetics; Tuberculosis;

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry by Kai On Chu; Thomas C. Ho; Wai Yee Chiang; Chi Chiu Wang; Dennis Shun Chiu Lam; Chi Pui Pang (199-204).
Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC–NCI–MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC–NCI–MS. The detection limit was 0.3 ng/ml, linearity over 0.995 from 0 to 300 ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300 ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200 ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000 ng/ml. We have provided an alternative, rapid, and robust method other than LC–MS–MS for TAA detection in AH.
Keywords: Triamcinolone acetonide; Aqueous humor; GC–NCI–MS;

Capacitively coupled contactless conductivity detection (C4D) is a new technique providing high sensitivity in capillary electrophoresis (CE) especially for small ions that can otherwise only be determined with indirect methods. In this work, direct determination and validation of valproic acid (VPA) in biological fluids was achieved using CE with C4D. VPA is of pharmacological interest because of its use in epilepsy and bipolar disorder. The running electrolyte solution used consisted of 10 mM 2-(N-morpholino)ethane sulfonic acid (MES)/dl-histidine (His) and 50 μM hexadecyltrimethylammonium bromide (HTAB) at pH 6.0. Caproic acid (CA) was selected as internal standard (IS). Analyses of VPA in serum, plasma and urine samples were performed in less than 3 min. The interference of the sample matrix was reduced by deproteinization of the sample with acetonitrile (ACN). The effect of the solvent type and ratio on interference was investigated. The limits of detection (LOD) and quantitation (LOQ) of VPA in plasma samples were determined as 24 and 80 ng/ml, respectively. The method is linear between the 2 and 150 μg/ml, covering well the therapeutic range of VPA (50–100 μg/ml).
Keywords: Valproic acid; Contactless conductivity detection; Capillary electrophoresis; Biological fluids; Caproic acid;

Determination of perospirone by liquid chromatography/electrospray mass spectrometry: Application to a pharmacokinetic study in healthy Chinese volunteers by Ning Ma; Wen-Ying Liu; Huan-De Li; Yun-Gui Zhu; Bi-Kui Zhang; Feng Wang; Rong-Hua Zhu; Da-Xiong Xiang (210-216).
Perospirone is a novel atypical antipsychotic with a unique combination of 5-HT1A receptor agonism as well as 5-HT2A and D2 receptor antagonism. A simple rapid and selective LC–MS method utilizing a single quadrupole mass spectrometer was developed and validated for the determination of perospirone hydrochloride in human plasma. N-hexane was used to extract perospirone hydrochloride and amlodipine benzenesulfonate (internal standard (IS)) from an alkaline plasma sample. LC separation was performed on a XTerra ® MS C18 column (100 mm × 2.1 mm, i.d. 3.5 μm) using methanol −10 mM ammonium acetate (84:16, v/v) as a mobile phase. The quantification of target compounds was obtained by using a selected ion monitoring (SIM) at m/z 427.5 [M + H]+ for perospirone hydrochloride, and at m/z 431.4 [M + Na]+ for IS (amlodipine benzenesulfonate). Perospirone and IS eluted as sharp, symmetrical peaks with retention times of 3.11 ± 0.01 min and 4.15 ± 0.2 min, respectively. Calibration curves of perospirone hydrochloride in human plasma at concentrations ranging from 0.10 to 21.1 ng/mL exhibited excellent linearity (r 2  = 0.9997). The mean absolute recovery of the drug from plasma was more than 85%. Intra- and inter-day relative standard deviations were less than 6.43% and 11.9% for perospirone hydrochloride at the range from 0.32 to 10.6 ng/mL. Stability characteristics of the drug-containing plasma were thoroughly evaluated to establish appropriate conditions to process, store and prepare for chromatographic analysis without inducing significant chemical degradation. The following pharmacokinetic parameters were elucidated after administering a single dose of 8 mg perospirone hydrochloride. The area under the plasma concentration versus time curve from time 0 to 24 h (AUC0–24) was 15.48 ± 4.23 μg/L h; peak plasma concentration (C max) was 2.79 ± 0.78 μg/L; time to C max (T max) was 1.79 ± 0.45 h; and elimination half-life (t 1/2) 6.78 ± 1.38 h. The described assay method showed acceptable precision, accuracy, linearity, stability, and specificity and can be used for pharmacokinetic studies, therapeutic drug monitoring, and drug abuse screening.
Keywords: Perospirone; Pharmacokinetic; LC-ESI/MS;

Solid-phase microextraction–liquid chromatography (SPME–LC) determination of fluoxetine and norfluoxetine in plasma using a heated liquid flow through interface by Christian Fernandes; Alvaro José dos Santos Neto; José Carlos Rodrigues; Claudete Alves; Fernando Mauro Lanças (217-223).
A simple and sensitive procedure using solid-phase microextraction coupled with high performance liquid chromatography (HPLC) to analyze fluoxetine (FLU) and its metabolite norfluoxetine (nor-FLU) in plasma samples was developed and validated. SPME conditions were optimized employing a factorial design. The sampling step was performed using a PDMS-DVB fiber and desorption was carried out in a novel homemade heated interface. Fluoxetine and norfluoxetine were analyzed by HPLC, using a C18 Phase Sep column (150 mm × 4.6 mm, 3 μm) packed “in house”, and acetonitrile:acetate buffer 25 mmol l−1 with triethylamine 25 mmol l−1 pH 4.6 (70:30) as the mobile phase. The developed method has shown precision, linearity, specificity, and limit of quantification (LOQ) adequate to assay fluoxetine and norfluoxetine in plasma. Furthermore, the results obtained using the homemade interface has shown an improvement in the desorption process when compared with the results obtained using the off-line mode.
Keywords: SPME; SPME–LC; Interface; Fluoxetine; Plasma;

Simultaneous determination of procaine and para-aminobenzoic acid by LC–MS/MS method by Mugunthu R. Dhananjeyan; Crystal Bykowski; Jill A. Trendel; Jeffrey G. Sarver; Howard Ando; Paul W. Erhardt (224-230).
A sensitive high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA). N-Acetylprocainamide (NAPA) was used as an internal standard for procaine and PABA analysis. This assay method has also been validated in terms of linearity, lower limit of detection, lower limit of quantitation, accuracy and precision as per ICH guidelines. Chromatography was carried out on an XTerra™ MS C18 column and mass spectrometric analysis was performed using a Quattro Micro™ mass spectrometer working with electro-spray ionization (ESI) source in the positive ion mode. Enhanced selectivity was achieved using multiple reaction monitoring (MRM) functions, m/z 237 → 100, m/z 138 → 120, and m/z 278 → 205 for procaine, PABA and NAPA, respectively. Retention times for PABA, procaine and NAPA were 4.0, 4.7 and 5.8 min, respectively. Linearity for each calibration curve was observed across a range from 100 nM to 5000 nM for PABA, and from 10 nM to 5000 nM for procaine. The intra- and inter-day relative standard deviations (RSD) were <5%.
Keywords: Esters; LC–MS/MS; Local anesthetics; NAPA; PABA; Procaine;

Practical HPLC methods for the quantitative determination of common antimalarials in Africa by D.J. Bell; S.K. Nyirongo; M.E Molyneux; P.A. Winstanley; S.A. Ward (231-236).
This article describes high-performance liquid chromatographic assays for the quantification of sulfadoxine (SDX), pyrimethamine (PYM), chloroquine (CQ), amodiaquine (AQ) and desethylamodiaquine (AQM) from whole blood. All four assays were set up and validated in Malawi using a common high-performance liquid chromatography platform and column and involved the use of simple mobile phase and extraction reagents. Calibration curves were linear (r 2  > 0.95) in the ranges 5–100 μg/ml, 50–1000, 150–1500, 100–1000 and 100–1000 ng/ml for SDX, PYM, CQ, AQ and AQM, respectively. Intra-assay and inter-assay coefficients of variation were <15% at 3 points spanning the concentration range and <20% at the lower limit of quantification. The assays were specific with no interference from the other antimalarials described in this report. All four assays use liquid–liquid extraction, reversed-phase chromatography and UV detection and require between 50 and 200 μl of blood. Because the assays share common instruments and reagents, they are cost-efficient and could be used to optimise antimalarial drug therapies in other resource poor settings.
Keywords: Sulfadoxine; Pyrimethamine; Chloroquine; Amodiaquine; Desethylamodiaquine; High-performance liquid chromatography; Malaria;

An accurate, rapid and simple liquid chromatography–tandem mass spectrometry (LC–MS–MS) assay method was developed for the determination of ziprasidone (ZIP) in the plasma of schizophrenia patients. A simple one step liquid–liquid extraction with 20% methylene dichloride in pentane was used to isolate ZIP and the internal standard from the plasma matrix. The compounds were separated on a C-18 column by an isocratic elution and the eluted compounds were analyzed by a triple quadrupole mass spectrometer with a TurboIon spray interface using the positive ion atmospheric pressure electrospray ionization method and detected using multiple reaction monitoring mode. The ZIP standard calibration curve was linear over the range of 0.25–500 ng/ml when 0.5 ml of plasma was used for the analysis (r 2  > 0.998). The intra-assay (within-day) and inter-assay (between-day) variations were less than 12% for the spiked standard curve and quality control samples. The absolute extraction efficiency was 82% for ZIP and 68% for INS-RSP. The analysis time for each sample was less than 3 min and useful for high turnaround plasma level determinations. This LC–MS–MS assay method for ZIP is highly specific, sensitive, accurate and rapid and is currently being used for the plasma level determination of ZIP in schizophrenia patients treated with various daily oral doses of ZIP. The data showed large inter-individual variations.
Keywords: Ziprasidone; LC–MS–MS; Schizophrenia patients; Plasma analysis; Ziprasidone levels;

In order to evaluate the differences in the partition properties of 35 structurally congeneric nucleobases of biological interests in octanol–water biphasic, alkyl C8/C18, and IAM systems, a comparative chromatographic study was performed. Comparing with the reversed-phase C8/C18 retention data, most of the purines possessed weaker IAM retention except for those with specific H-bond and/or electrostatic interactions. Quantitative correlations between the experimental log P ow literature values and the IAM, C8, and C18 log k were evaluated (R 2  = 0.943, 0.794, and 0.767, respectively). Although IAM retention correlated significantly better (larger R 2 value) with the log P ow values statistically, the latter was revealed apparently behaving more like (slope approaching unity) alkyl C8/C18 retention and hence also has the same shortcoming in under-representing analytes capable of forming short-term H-bond/electrostatic interactions with polar head-groups of phospholipids. A chemically meaningful structure-retention model (q 2  = 0.824 and R 2  = 0.968) was derived, in which the hydrophobic interaction is identified as the underlying factor for the retention of purines in IAM system modulated non-trivially by H-bond/electrostatic interactions.
Keywords: Nucleobases; Retention; Partition; IAM; C8/C18; Octanol–water log P;

Response surface methodology for the evaluation of glucose-6-phosphate dehydrogenase enrichment process by soybean lecithin reversed micelles by Francislene Andréia Hasmann; Daniela de Borba Gurpilhares; Inês Conceição Roberto; Adalberto Pessoa (262-266).
Glucose-6-phosphate dehydrogenase (G6PD) present in Saccahromyces cerevisiae is an enzyme of the pentose pathway. An effective enrichment of this intracellular enzyme can be achieved with the reversed micellar methodology. In this work, this methodology was employed with soybean lecithin, a biocompatible surfactant. A factorial design was used to evaluate the influence of pH (A) and extraction runs (B) on the G6PD purification factor. After statistical analysis and process optimization, a mathematical model representing G6PD enrichment was obtained: Y  = 4.89 − 0.83A  + 0.092B  + 0.27AB  − 1.37B2 with an enzyme purification factor of about 5.2.
Keywords: Reversed micelles; Soybean lecithin; Response surface methodology; Glucose-6-phosphate dehydrogenase;

A method for urinary peptide(s) and protein hydrolysis, involving autoclaving at 15 psi (121 °C) for 60 min, is described. Using three candidate proteins (bovine serum albumin, casein and gelatin) and urine specimens, the effect of autoclaving with respect to the optimum time required for hydrolysis under both acidic (6 N HCl) and alkaline (6 N KOH) conditions was studied. Recoveries of total amino acids from proteins and urine hydrolysate(s) suggest that complete hydrolysis of proteins and urinary peptides could be achieved by autoclaving for 30–60 min instead of 16 h of incubation at 110 °C. Further, stability of some of the individual amino acids was also studied. The observed differential stability of amino acids under acidic and alkaline conditions, as demonstrated in this study by HPLC analysis, makes it imperative to choose the appropriate hydrolytic condition while studying the composition of any given amino acids in urinary peptide(s)/protein hydrolysates. Further, the finding that both Pro and Hyp were stable under alkaline conditions of hydrolysis by autoclaving renders this method suitable for assaying these two amino acids from urine hydrolysates, hence its utility in the study of urinary peptide derived Hyp and Pro in bone/cartilage disorders.
Keywords: Autoclave; Proteins; Urinary peptides; Prolyl residues; Hydrolysis; Bone disorders;

This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1′-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography–mass spectrometry (LC–MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1 ml of alkalinized plasma using n-hexane–chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C18, 50 mm × 2.0 mm i.d.). The mobile phase for separation consisted of 10 mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2 ml/min. The drift voltage was 100 V. The sampling aperture was heated at 120 °C and the shield temperature was 260 °C. The total time for chromatographic separation was less than 16 min. The validated concentration ranges of this method were 0.25–50 ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40 ng/ml. The limits of quantification were 0.25 ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1 mg) and a single-oral administration (2 mg) of subtherapeutic MDZ dose.
Keywords: Midazolam; 1′-Hydroxymidazolam; CYP3A; LC–MS;

Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry by Tobias Langrock; Natividad García-Villar; Ralf Hoffmann (282-288).
Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4-cis-/trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N 2-(5-fluoro-2,4-dinitrophenyl)-l-valine amide (l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.
Keywords: Electrospray; FDVA; Hydroxylation; Hyl; Hyp; RP-HPLC;

Development and validation of an immunochromatographic assay for rapid detection of sulfadiazine in eggs and chickens by Xiliang Wang; Kui Li; Deshi Shi; Xiue Jin; Ning Xiong; Fuhu Peng; Dapeng Peng; Dingren Bi (289-295).
A rapid immunochromatographic assay (ICA) was developed and validated for the detection of sulfadiazine in eggs and chickens. Based on the competitive reaction mechanism, the competitor of sulfadiazine (sulfadiazine–BSA conjugate) was immobilized to the defined detection zone on a nitrocellulose membrane which acted as the capture reagent, and the monoclonal antibody against sulfadiazine was conjugated to colloidal gold particles which served as the detection reagent for the preparation of the immunochromatographic strips to test sulfadiazine. With this method, the semi-quantitative detection of sulfadiazine was accomplished in less than 15 min, with high sensitivity to sulfadiazine (5 ng/g) and low cross-reactivities with other sulfonamides. With experimental egg and chicken samples spiked with sulfadiazine at concentrations of 10, 20, and 100 ng/g, recoveries were demonstrated to be from 71% to 97% in egg samples and 71% to 95% in chicken samples. This method was compared with the enzyme-linked immunosorbent assay by testing 52 egg samples from the animal experiment, and compared with the high-performance liquid chromatographic method by testing 56 chicken samples, with an agreement rate of 100% for both comparisons, by using the maximum allowed residue of sulfadiazine (i.e. 100 ng/g) as the cut-off level as set by the European Union and China. The accuracy of ICA was also confirmed in an initial study with marketed egg and chicken samples. In conclusion, the method is rapid and accurate for the detection of sulfadiazine in eggs and chickens.
Keywords: Immunochromatographic assay; Colloidal gold; Strip; Sulfadiazine; Egg; Chicken;

A rapid, sensitive and selective LC–MS method is described for the simultaneous determination of zuclomiphene and enclomiphene in plasma from patients undergoing treatment with clomiphene citrate for the induction of ovulation. Samples spiked with N-didesmethyltamoxifen, the internal standard, were extracted into methyl tertiary butyl ether. The compounds were separated on a Luna C18 analytical column, and a mobile phase of methanol–water (70:30 v/v) containing 0.05% trifluoroacetic acid at a flow rate of 1 ml/min. The limits of determination were 35 pg/ml and 7 pg/ml for zu- and enclomiphene, respectively. Within-day coefficients of variation ranged from 2.1% to 7.2%.
Keywords: Clomiphene citrate; Zuclomiphene; Enclomiphene;

Capillary electrophoretic assay for nitrate levels in the vitreous of proliferative diabetic retinopathy by Leyi Gao; Jose S. Pulido; R. Mark Hatfield; Robert F. Dundervill; Colin A. McCannel; Scott A. Shippy (300-304).
The determination of nitric oxide (NO) in human vitreous samples is complicated by the relatively short half-life of the analyte and the viscous, high salt and protein biological matrix. In this work, we developed a fast (<5 min) and useful CE method to determine the stable metabolite, nitrate, from vitreous samples. This proposed method has been successfully applied to determine the nitrate levels from the vitreous humor of patients undergoing vitrectomy for a variety of conditions. A statistically significant increase (P  = 0.000001) of the mean level of nitrate was observed in vitreous humor of patients with proliferative diabetic retinopathy (41.17 ± 4.09 μM, n  = 27) versus controls (15.22 ± 0.86 μM, n  = 35). The elevated levels of nitrate in the vitreous of patients known to have diabetic retinopathy suggests that NO is involved with the pathology of this disease.
Keywords: Nitric oxide; PDR; Capillary electrophoresis; Clinical samples;

Statistical power and analytical quantification by Pedro Araujo; Livar Frøyland (305-308).
It is suggested that power analysis should be formally incorporated into quantification experiment reports in order to substantiate the conclusions derived from experimental data more effectively. The article addressed the issues of power analysis calculation, sample size estimation and appropriate data reporting in quantitative analytical comparisons. Illustrative examples from the literature are used to show how the described power analysis theory could be applied in practice.
Keywords: Statistical power; Analytical comparisons; Quantification; Validation; Chromatographic methods;