Journal of Chromatography B (v.862, #1-2)

This review provides the achievements of enantioseparation of adrenergic drugs and application of these methods in clinical and pharmaceutical analysis. The adrenergic agonist and antagonist drugs are analyzed in the direct and indirect modes by liquid chromatography (LC) and capillary electrophoresis (CE). Other chromatographic enantioseparation methods including super- and sub-critical fluid chromatography (SFC), and capillary electrochromatography (CEC) are presented likewise to analyse the cited compounds. The different separation processes for these drugs are briefly discussed and some applications are presented.
Keywords: Enantioseparation; Adrenergic drugs; Agonists; Antagonists; Liquid chromatography; Capillary electrophoresis;

Analytical methods applied to the determination of heterocyclic aromatic amines in foods by M. Sanz Alaejos; J.H. Ayala; V. González; A.M. Afonso (15-42).
Analytical aspects concerning the heterocyclic aromatic amines (HAAs) determination in foods are reviewed. Sample pre-treatment procedures such as liquid–liquid extraction (LLE), supercritical fluid extraction, solid-phase extraction (SPE), solid-phase microextraction (SPME), and the mainly used LLE–SPE tandem extraction are discussed. The analytical methods used for the identification and quantification are HPLC, HPLC combined with single or tandem MS detection (HPLC-MS, HPLC-MS/MS), GC–MS and capillary electrophoresis. Advantages and figures of merit for each technique are discussed.
Keywords: Heterocyclic aromatic amines; Foods; Extraction methods; Chromatographic methods/mass spectrometry; Capillary electrophoresis;

A sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA–Na2–5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 μg g−1 dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 μg g−1 DW, with a limit of quantification (LOQ) of 0.02 μg g−1 DW. The limit of detection (LOD) of the method was 0.007 μg g−1 DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR.
Keywords: Microcystin-LR; Glutathione conjugate; LC–MS; Quantitative determination;

A novel preparative HPLC method separating silybin has been developed to meet the need for both silybin A and silybin B standard. After the preparation of silybin A and silybin B standard, a simple, sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry (LC–MS–MS) method with negative electrospray ionization (ESI) was developed for the quantification of silybin A and silybin B in human plasma. Following rapid sample preparation, silybin A, silybin B and naringin (internal standard, ISTD) were separated on a Zorbax Eclipse XDB-C18 column, using methanol–water containing 0.1% formic acid (48:52, v/v) as the mobile phase. The mass spectrometer was operated in selected reaction monitoring (SRM) mode using the transition m/z 481.1 → 300.9 for both silybin A and silybin B and m/z 579.2 → 271.1 for naringin, respectively. Linear calibration curves were obtained in the concentration range of 2–5000 ng/ml with a lower limit of quantitation (LLOQ) of 2 ng/ml for both silybin A and silybin B, respectively. The intra- and inter-day precision values were below 7.5% and accuracy was within ±4.9% at all three quality control (QC) levels, for both silybin A and silybin B, respectively. This method was successfully applied to the stereospecific analysis of silybin in plasma samples from a pharmacokinetic study of silybin A and silybin B in 22 healthy male Chinese volunteers after a single oral dose of silybin–phosphatidylcholine complex (equivalent to 280 mg silybin, including 133 mg silybin A and 147 mg silybin B).
Keywords: Pre-HPLC; LC–MS–MS; Silybin; Pharmacokinetic; Silybin A; Silybin B; Plasma;

Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles by E. Feitosa; K.T. Catelam; F.A. Hasmann; Hans-Olof Johansson; I.C. Roberto; A. Pessoa (58-63).
A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes.
Keywords: Phase diagram; Liquid–liquid extraction; Reverse micelles; CTAB; Enzymes; Glucose-6-phosphate dehydrogenase; Xylose redutase;

A simple and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method using an atmospheric pressure chemical ionization source (APCI) for the quantification of fenretinide (4-HPR) in mouse plasma was developed and validated. After a simple protein precipitation of plasma sample by acetonitrile, 4-HPR was analyzed by LC–APCI–MS/MS. High-performance liquid chromatography (HPLC) separation was conducted on a Hypurity C18 column (50 mm × 2.1 mm, 5 μm) with a flow rate 0.60 mL/min using a gradient mobile phase comprised of 0.05% formic acid in water (A) and methanol (B), and a run time of 4.5 min. The elimination of a tedious sample preparation process and a shorter run time substantially reduced total analysis time. The method was linear over the range 0.5–100 ng/mL, with r  > 0.998. The intra- and inter-assay precisions were 1.4–9.2% and 5.1–8.2%, respectively, and the intra- and inter-assay accuracies were 93.9–98.6% and 92.7–95.3%, respectively. The absolute recoveries were 90.3% (1.5 ng/mL), 97.0% (7.5 ng/mL) and 92.1% (75.0 ng/mL) for 4-HPR, and 99.1% for the internal standard (150 ng/mL). The analytical method had excellent sensitivity using a small sample volume (30 μL) with the lower limit of quantification (LLOQ) 0.5 ng/mL. This method is robust and has been successfully employed in a pharmacokinetic study of 4-HPR in a mouse xenograft model of neuroblastoma.
Keywords: Fenretinide (4-HPR); N-(4-Methoxyphenyl) retinamide (4-MPR); LC–APCI–MS/MS; Plasma;

A sensitive and rapid liquid chromatography–mass spectrometric method for the simultaneous determination of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat plasma was developed and validated. Chromatographic separation was performed on a C18 column using a step gradient program with the mobile phase of 0.5 mmol/L ammonium chloride solution and acetonitrile. The analytes and I.S. were detected using an electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. The method was linear over the investigated concentration range with a good correlation coefficient higher than 0.997. The lower limits of detection (LLOD) of these analytes were all lower than 2.0 ng/mL. The intra- and inter-day precisions were all no more than 7.5% and accuracies were within the range of 97.5–107.0%. The validated method was successfully applied to investigate the pharmacokinetics of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat after intravenous administration of ‘SHENMAI’ injection.
Keywords: Ginsenoside Rg1; Re; Rd; Rb1; Ophiopogonin D; LC–ESI-MS; Pharmacokinetics; ‘SHENMAI’ injection;

A capillary electrophoresis method was developed for the enantioselective quantification of methadone (MTD) and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP). The enantiomers of MTD and EDDP were resolved by CE in 5 min using 0.2% highly sulphated gamma-cyclodextrins as chiral selectors and a 50 mM phosphate solution at pH 4.5 as background electrolyte. The optimized method was applied and validated for oral fluid testing. Linear relationships were obtained for MTD enantiomers in the range of 8.1–625 ng/mL and in the range of 7.6–500 ng/mL for EDDP enantiomers. The detection limits ranged from 2.3 to 2.4 ng/mL, whereas the limits of quantification ranged from 7.6 to 8.1 ng/mL. Intra- and inter-assay precision and accuracy were acceptable, respectively. The method was applied to the analyses of 60 oral fluid specimens obtained from patients enrolled in a MTD maintenance programme. Our data pointed out that higher concentrations of (R)-MTD and the enantioselective excess of (S)-EDDP in OF may reflect the free fraction of MTD and EDDP enantiomers in plasma.
Keywords: Oral fluid; Methadone; 2-Ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine; Capillary electrophoresis; Highly sulphated γ-cyclodextrin;

A rapid, sensitive and reproducible gas chromatographic method with flame ionization detection is described for the simultaneous identification and quantification of 33 amino acids and dipeptides in spent cell culture media in under seven minutes. The method involves the use of the EZ:faast™ (Phenomenex) amino acid sample testing kit. Instrumental and assay precision, percent recovery, linear range, limit of detection and peak identity in highly complex cell culture media containing either soy hydrolysate or fetal bovine serum were validated using gas chromatography-flame ionization detector (GC-FID).
Keywords: Spent cell culture media; Amino acids derivatives; Dipeptide derivatives; EZ:faast kit; Liquid phase extraction; Solid phase extraction; Gas chromatography-flame ionization detector;

The objective of this study was to develop a procedure for the GC/MS assay of paraquat in meconium as a biomarker of fetal exposure to paraquat. The method involved a sodium borohydride-nickel chloride reduction procedure, liquid–liquid extraction of the perhydrogenated product, concentration, and GC/MS assay. The method demonstrated good overall recovery (102.56%) with %CV (inter-assay) of less than 13%, and a limit of detection of 0.0156 μg/g. Analysis of meconium samples from a study population in the Philippines (n  = 70) showed a 2.8% prevalence of fetal exposure to paraquat.
Keywords: Paraquat; Meconium; Meconium analysis; GC/MS;

A quantitative method using liquid chromatography–tandem mass spectrometry (LC–MS–MS) was developed for the simultaneous determination of 23 endogenous steroids in primate urine. The introduced method includes estrone, pregnandiol, cortisol, testosterone and several human urinary glucocorticoid and androgen metabolites. As the method is intended for the analysis of steroid hormones in behavioral studies on wild-living primates, it was adapted for a sample volume of 200 μL urine. The sample preparation consisted of an enzymatic hydrolysis of steroid glucuronides using β-glucuronidase from E. coli followed by a solvolytic cleavage of steroid sulfates employing sulfuric acid/ethyl acetate. The extraction of steroids from urine was optimized with respect to pH during extraction, type of ether and the amount of enzyme necessary for complete hydrolysis of glucuronides. The recovery of steroids spiked into urine before hydrolysis was 58.9–103.7% with an intra-day precision of 2.7–14.3% and an inter-day precision of 2.9–14.8%. Detection limits ranged from 0.1–0.5 ng/mL. The reproducibility of the whole sample preparation process was also demonstrated for unspiked urine (CV 1.2–16.5%). The proportion of steroid hormone excreted as sulfate was determined for 21 steroids in chimpanzee urine. The solvolysis proved to be essential for all investigated steroids except for pregnandiol, tetrahydrocortisol and tetrahydrocortisone, which were found to be less then 10% in the solvolysis fraction.
Keywords: Primate urine; Cortisol; Testosterone; Estrone; Pregnandiol; Endogenous steroids; Liquid chromatography–mass spectrometry; Solvolysis; Steroid sulfates;

Determination of aldosterone in serum by liquid chromatography–tandem mass spectrometry by Ursula Turpeinen; Esa Hämäläinen; Ulf-Håkan Stenman (113-118).
Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography–mass spectrometry (ID-GC–MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography–mass spectrometry (LC–MS) is a potentially superior method. The analysis utilizes 0.5 mL of serum. The samples were extracted with dichloromethane–ether. The extract was evaporated to dryness and aldosterone was analyzed by LC–MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60–3000 pmol/L. Interassay CVs were 4.3–7.5% at aldosterone concentrations of 97–993 pmol/L. The lower limit of quantitation (LOQ) was 30 pmol/L (signal to noise ratio = 10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC–MS/MS (x) and RIA (y) method was: y  = 1.33x  + 185 (r  = 0.95; n  = 124). Sensitivity and specificity of the LC–MS/MS method for serum aldosterone offer advantages over GC–MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC–MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC–MS/MS.
Keywords: Serum aldosterone; LC–MS/MS; ESI; Electrospray ionization mass spectrometry; API 3000; RIA;

Simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry by Yan Yang; Hao Li; Kan Gao; Mingyuan Liu; Yantong Sun; Tingting Yan; J. Paul Fawcett; Yimin Cui; Jingkai Gu (119-124).
A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma was developed. After sample preparation by protein precipitation and liquid–liquid extraction, the analytes and internal standard (IS) were separated on a Venusil XBP-C8 column using gradient elution. Multiple reaction monitoring of dexamethasone palmitate, dexamethasone and IS used the precursor to product ion transitions at m/z 631.8 → 373.1, m/z 393.2 → 147.1 and m/z 264.2 → 58.1, respectively. The method was linear over the ranges 1.5–1000 ng/mL for dexamethasone palmitate and 2.5–250 ng/mL for dexamethasone with intra- and inter-day precisions of <10% and accuracies of 100 ± 7%. The assay was applied to a clinical pharmacokinetic study involving the injection of dexamethasone palmitate to healthy volunteers.
Keywords: Dexamethasone; Dexamethasone palmitate; LC/MS/MS; Pharmacokinetics; Human;

HPLC fingerprinting and LC–TOF-MS analysis of the extract of Pseudostellaria heterophylla (Miq.) Pax root by Chao Han; Yan Shen; Junhui Chen; Frank Sen-Chun Lee; Xiaoru Wang (125-131).
High-performance liquid chromatographic (HPLC) was developed for fingerprint analysis of Pseudostellaria heterophylla (Miq.) Pax. Liquid chromatography–electrospray ionization-time-of-flight mass spectrometry (LC–TOF-MS) technique was first employed to identify the components of the fingerprint. Twelve major peaks in chromatographic fingerprint were analyzed by on-line LC–TOF-MS analysis; one cyclic peptide was unequivocally identified and five cyclic peptides were tentatively assigned based on their MS data. These cyclic peptides served as the marker peaks in the HPLC fingerprints. The chromatographic fingerprints have been analyzed by similarity index calculations and hierarchical clustering analysis (HCA). The result showed that the HPLC fingerprints could be used to determine the optimal harvest time for P. heterophylla (Miq.) Pax and to authenticate the species of the herb.
Keywords: Fingerprint chromatogram; Pseudostellaria heterophylla (Miq.) Pax; HPLC–TOF-MS; Hierarchical clustering analysis; Similarity analysis; Cyclic peptides;

Determination of levocetirizine in human plasma by liquid chromatography–electrospray tandem mass spectrometry: Application to a bioequivalence study by M.R. Morita; D. Berton; R. Boldin; F.A.P. Barros; E.C. Meurer; A.R. Amarante; D.R. Campos; S.A. Calafatti; R. Pereira; E. Abib; J. Pedrazolli (132-139).
We describe a liquid chromatography–tandem mass spectrometric method (LC–MS/MS) for levocetirizine quantification (I) in human plasma. Sample preparation was made using a fexofenadine (II) addition as internal standard (IS), liquid–liquid extraction using cold dichloromethane, and dissolving the final extract in acetonitrile. I and II (IS) were injected in a C18 column and the mobile phase composed of acetonitrile:water:formic acid (80.00:19.90:0.10, v/v/v) and monitored using positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 389 > 201 for I and m/z 502 > 467 for II. The limit of quantification and the dynamic range achieved were 0.5 ng/mL and 0.5–500.0 ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples taken up to 48 h after oral administration of 5 mg of levocetirizine dichloridrate in healthy volunteers demonstrate its applicability to bioavailability studies.
Keywords: Mass spectrometry; Bioavailability; Levocetirizine;

Enantiospecific gas chromatographic–mass spectrometric analysis of urinary methylphenidate: Implications for phenotyping by Natalie L. LeVasseur; Hao-Jie Zhu; John S. Markowitz; C. Lindsay DeVane; Kennerly S. Patrick (140-149).
A chiral derivatization gas chromatographic–mass spectrometric (GC–MS) method for urine methylphenidate (MPH) analysis was developed and validated to investigate preliminary findings regarding a novel MPH poor metabolizer (PM). Detection was by electron impact (EI) ionization-selected ion monitoring of the N-trifluoroacetylprolylpiperidinium fragments from MPH and the piperidine-deuterated MPH internal standard. The PM eliminated ∼70 times more l-MPH in urine (9% of the dose over 0–10 h), and ∼5 times more of the d-isomer (10% of the dose), than the mean values determined from 10 normal metabolizers of MPH. Only minor amounts of the metabolite p-hydroxy-MPH were found in the urine of both the PM and normal metabolizers, while the concentration of MPH lactam was not high enough to be detectable. The described method indirectly gauges the functional carboxylesterase-1 status of patients receiving MPH based on the evaluation of relative urine concentrations of d-MPH:l-MPH. Clinical implications concerning rational drug selection for an identified or suspected MPH PM are discussed.
Keywords: Methylphenidate; Esterase; Pharmacogenetics; Enantiomers; Urine;

A simple, reliable and sensitive liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for quantification of N-acetylglucosamine in human plasma. Plasma samples were pretreated with acetonitrile for protein precipitation. The chromatographic separation was performed on Hypersil Silica column (150 mm × 2 mm, 5 μm). The deprotonated analyte ion was detected in negative ionization mode by multiple reaction monitoring mode. The mass transition pairs of m/z 220.3 → 118.9 and m/z 226.4 → 123.2 were used to detect N-acetylglucosamine and internal standard 13C6-N-acetylglucosamine, respectively. The assay exhibited a linear range from 20 to 1280 ng/ml for N-acetylglucosamine in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully applied to analyze human plasma samples in a pharmacokinetic study.
Keywords: LC–MS/MS; N-Acetylglucosamine; Pharmacokinetic study;

Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH2 domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography.
Keywords: Recombinant monoclonal antibody; Oligosaccharides; WCX-10; Mass spectrometry;

Quantitative determination of cyclic phosphatidic acid in human serum by LC/ESI/MS/MS by Lian Shan; Shanping Li; Keeve Jaffe; Lorelei Davis (161-167).
An LC/ESI/MS/MS method for cyclic phosphatidic acid (cPA) quantification in serum is established in the present report. The limit of quantitation of the assay reaches low nanomolar level in human serum and the CV% are within 10%. Using this method, we successfully quantify the levels of two cPA species, 16:0 and 18:1, in human serum. We find that the concentrations of 16:0 cPA in the serum of normal subjects and post-surgery ovarian cancer patients are significantly higher than its corresponding concentration in pre-surgery ovarian cancer patients, supporting the observation that cPA has anti-cancer activity. Another discovery is that the addition of strong acids (such as hydrochloric acid) in human serum may lead to the production of artificial cPA. Therefore, strong acids should be avoided in the extraction of cPA present in a complex matrix. Based on this observation, a new lipid extraction method was developed and used to extract cPA. The extraction recovery is close to 80%, guaranteeing an accurate quantification of cPA by LC/ESI/MS/MS can be performed.
Keywords: Cyclic phosphatidic acid (cPA); cPA quantification; LC/ESI/MS/MS;

Liquid chromatography–mass spectrometric assay for quantitation of the short-chain fatty acid, 2,2-dimethylbutyrate (NSC 741804), in rat plasma by Robert A. Parise; Jan H. Beumer; Cyrous O. Kangani; Julianne L. Holleran; Julie L. Eiseman; Nicola F. Smith; Joseph M. Covey; Susan P. Perrine; Merrill J. Egorin (168-174).
2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To facilitate pharmacokinetic evaluation of DMB, we developed an LC–MS assay and quantitated DMB in plasma of rats after an oral dose of 500 mg/kg. After acetonitrile protein precipitation, DMB and dimethylvaleric acid (DMV) internal standard were derivatized to benzylamides, chromatographed on a Hydro-RP column with acetonitrile, water, and 0.1% formic acid, and detected by electrospray positive-mode ionization mass spectrometry. The assay was accurate (97–107%) and precise (3.4–6.2%) between 100 and 10,000 ng/mL. Recovery from plasma was >62%. Plasma freeze–thaw and room temperature stability were acceptable.
Keywords: Dimethylbutyrate; Metabolism; Mass spectrometry; Validation; Fatty acid; Sickle cell disease; Thalassemia;

A sensitive high-performance liquid chromatographic method for determination of ranitidine (RAN) in rabbit plasma is described. The method is based on liquid–liquid extraction, labeling with dansyl chloride and monitoring with fluorescence detector at 338 nm (ex)/523 nm (em). Plasma samples were extracted with diethyl ether alkalinized with 1 M sodium hydroxide. Ephedrine HCl (EPH-HCl) was used as internal standard. Both, RAN and EPH were completely derivatized after heating at 60 °C for 10 min in sodium bicarbonate solution (pH 9.5). The derivatized samples were analyzed by HPLC using Agilent Zorbax Extended C18 column (150 mm × 4.6 mm i.d.) and mobile phase consists of 48% acetonitrile and 52% sodium acetate solution (0.02 M, pH 4.6). The linearity of the method was in the range of 0.025–10 μg/ml. The limits of detection (LOD) and quantification (LOQ) were 7.5 ± 0.18 and 22.5 ± 0.12 ng/ml, respectively. Ranitidine recovery was 97.5 ± 1.1% (n  = 6; R.S.D. = 1.8%). The method was applied on plasma collected from rabbits at different time intervals after oral administration of 5 mg/kg ranitidine HCl.
Keywords: Ranitidine; Ephedrine; Dansyl chloride; Plasma; Fluorescence; HPLC;

In-tube solid-phase microextraction coupled to liquid chromatography (in-tube SPME/LC) analysis of nontricyclic antidepressants in human plasma by Bruno José Gonçalves Silva; Fernando Mauro Lanças; Maria Eugênia Costa Queiroz (181-188).
A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for simultaneous determination of mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline in human plasma was developed, validated and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. The quantification limits of the in-tube SPME/LC method varied between 20 and 50 ng/mL, with a coefficient of variation lower than 10%. The response of the in-tube SPME/LC method for most of the drugs was linear over a dynamic range from 50 to 500 ng/mL, with correlation coefficients higher than 0.9985. The in-tube SPME/LC can be successfully used to analyze plasma samples from ageing patients undergoing therapy with nontricyclic antidepressants.
Keywords: In-tube solid-phase microextraction; Plasma samples; Nontricyclic antidepressants;

Determination of CQP propionic acid in rat plasma and study of pharmacokinetics of CQP propionic acid in rats by liquid chromatography by Chang-hui Liu; Xiao-tao Huang; Rong Zhang; Lei Yang; Tian-lai Huang; Ning-sheng Wang; Sui-qing Mi (189-195).
A sensitive method for the determination of CQP propionic acid in rat plasma was developed and validated after solid-phase extraction. Chromatographic separation was achieved on a reversed-phase Alltima C18 column with the mobile phase of methanol–0.15% (v/v) phosphoric acid solution (pH 2.5) and step gradient elution resulted in a total run time of about 20 min. The analytes were detected by using UV detector at 345 nm. A good linear relationship was obtained in the concentration range of 50–12,800 ng/mL (r  = 0.9998). The intra-day RSDs and the inter-day RSDs at the concentration of 200, 800, 6400 and 12,800 ng/mL were less than 7.0% and 11.0%, respectively. The intra-day accuracy ranged from 96.3 to 106.5% and the inter-day accuracy ranged from 98.6 to 113.4%, respectively. Average extraction recoveries ranged from 83.6 to 94.3% in plasma at the concentrations of 200, 800, 6400 and 12,800 ng/mL. This method was successfully applied to the pharmacokinetic studies on rats.
Keywords: CQP propionic acid; Pharmacokinetics; Liquid chromatography;

High-throughput screening (HTS) for pharmaceutical leads requires sufficient number of samples with vast chemical diversity. In this paper, we proposed Chinese herbal formulas as an attractive source for HTS and introduced a strategy for the production of high-quality fractionated libraries. An offline two-dimension liquid chromatography protocol was developed to separate medium- and low-polar extract (MLPE) of Chinese herbal formulas, which implemented the production of semi-purified mixture libraries. HPLC coupled with diode-array detector (DAD) and mass spectrum (MS) analysis was performed to obtain MS and UV spectrum of library components. The detected components were characterized by retention, molecular weight and UV absorbance assisted by WiseProcessor, a customer-developed software to automatically process analytical data. Based on the current understanding in pathophysiology and pharmacology, multiple cell-based bioassays were performed to screening the library samples. Through validation and dereplication process, bioactive compounds could be identified rapidly. The combination of off-line two-dimension liquid chromatography separation, HPLC-DAD-MS analysis and computer-aided data processing is reliable and efficient for the utilization of Chinese herbal formulas as valuable sources for HTS. As a demonstration, a library sample set was generated from Qi-Xue-Bing-Zhi Formula, an efficient Chinese herbal formula to treat atherosclerosis. Several bioactive compounds were quickly identified from this library through the screening and dereplication process.
Keywords: Chinese herbal formulas; Fractionated library; HPLC-DAD-MS; High-throughput screening; Medium- and low-polar extract;

Determination of colistin in human plasma, urine and other biological samples using LC–MS/MS by Zheng Ma; Jiping Wang; Jacobus P. Gerber; Robert W. Milne (205-212).
A liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed to quantify colistin in human plasma and urine, and perfusate and urine from the isolated perfused rat kidney (IPK). Solid phase extraction (SPE) preceded chromatography on a Synergi Fusion-RP column with a mobile phase of acetonitrile, water and acetic acid (80/19/1) at 0.2 mL/min. Ions were generated using electrospray ionization and detected in the positive-ion mode. Multiple reaction monitoring was performed using precursor-product ion combinations. Calibration curves were linear from 0.028 μg/mL (human plasma, IPK perfusate and urine)/0.056 μg/mL (human urine) to 1.78 μg/mL (all four media) for colistin A sulfate; corresponding values for colistin B sulfate were 0.016/0.032 to 1.01 μg/mL. Accuracy and precision were within 10%. The LLOQ for colistin A sulfate was 0.028 μg/mL in human plasma, IPK perfusate and urine and 0.056 μg/mL in human urine; corresponding values for colistin B sulfate were 0.016 and 0.032 μg/mL. The low sample volume, short analysis time and low LLOQ are ideal for pre-clinical and human pharmacokinetic studies of colistin.
Keywords: Colistin; LC–MS/MS; SPE; Plasma; Urine;

A simple, rapid liquid chromatography/tandem mass spectrometric (LC–MS/MS) assay was developed and validated for the quantification of both unbound and total paclitaxel in plasma following treatment with Abraxane (ABI-007) or Taxol. Accurate and reproducible analysis of ABI-007, an albumin nanoparticle formulation of paclitaxel could not be achieved using previously published methodology designed for Taxol. The final validated method involved protein precipitation followed by vacuum filtration, in a 96-well format for rapid processing. The 4 min run employed gradient elution on a Waters SymmetryShield C8 (2.1 mm × 50 mm, 3.5 μm) column, followed by tandem mass spectrometric detection, in electrospray positive mode. Calibrator samples were prepared daily with paclitaxel and analyzed with both ABI-007 and paclitaxel quality control samples. To measure unbound drug, sample preparation was preceded by ultrafiltration. The assay was linear over the range of 10–2500 ng/mL, with dilution providing measurement up to 50,000 ng/mL. Within-run and between-run precision for all QC samples was less than 5.0% and 10.4%, respectively. Accuracy was high, with deviation of less than 6.1% for all QCs. Measurement of unbound paclitaxel was precise (BRP and WRP <10%).
Keywords: Albumin; Nanoparticle; Paclitaxel; Taxane; Anticancer; Formulation; LC–MS/MS; Unbound fraction; Free fraction;

A fully automated plasma protein precipitation sample preparation method for LC–MS/MS bioanalysis by Ji Ma; Jianxia Shi; Hoa Le; Robert Cho; Judy Chi-jou Huang; Shichang Miao; Bradley K. Wong (219-226).
This report describes the development and validation of a robust robotic system that fully integrates all peripheral devices needed for the automated preparation of plasma samples by protein precipitation. The liquid handling system consisted of a Tecan Freedom EVO® 200 liquid handling platform equipped with an 8-channel liquid handling arm, two robotic plate-handling arms, and two plate shakers. Important additional components integrated into the platform were a robotic temperature-controlled centrifuge, a plate sealer, and a plate seal piercing station. These enabled unattended operation starting from a stock solution of the test compound, a set of test plasma samples and associated reagents. The stock solution of the test compound was used to prepare plasma calibration and quality control samples. Once calibration and quality control samples were prepared, precipitation of plasma proteins was achieved by addition of three volumes of acetonitrile. Integration of the peripheral devices allowed automated sequential completion of the centrifugation, plate sealing, piercing and supernatant transferral steps. The method produced a sealed, injection-ready 96-well plate of plasma extracts. Accuracy and precision of the automated system were satisfactory for the intended use: intra-day and the inter-day precision were excellent (C.V. < 5%), while the intra-day and inter-day accuracies were acceptable (relative error < 8%). The flexibility of the platform was sufficient to accommodate pharmacokinetic studies of different numbers of animals and time points. To the best of our knowledge, this represents the first complete automation of the protein precipitation method for plasma sample analysis.
Keywords: Fully automated; Plasma; Sample preparation; LC–MS/MS; Bioanalysis;

A bioanalytical method for the analysis of piperaquine in human plasma using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. It was found that a mobile phase with high pH (i.e. 10) led to better sensitivity than mobile phase combinations with low pH (i.e. 2.5–4.5) despite the use of positive electrospray and a basic analyte. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as R.S.D., were lower than 7% at all tested concentrations (4.5, 20, 400 and 500 ng/mL) and below 10% at the lower limit of quantification (LLOQ) (1.5 ng/mL). The calibration range was 1.5–500 ng/mL with a limit of detection (LOD) at 0.38 ng/mL. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. Matrix effects originating from the sample clean-up (i.e. solid-phase extraction) procedure rather than the plasma background were responsible for the ion suppression seen in this study. Salts remaining from the buffers used in the solid-phase extraction suppressed the signals for both piperaquine and its deuterated internal standard. This had no effect on the quantification of piperaquine. Triethylamine residues remaining after evaporation of the solid-phase extraction eluate were found to suppress the signals for piperaquine and its deuterated internal standard differently. It was found that this could lead to an underestimation of the true concentration with 50% despite the use of a deuterated internal standard.
Keywords: Antimalarial; Differential matrix effect; High throughput; Ion suppression; Liquid chromatography/tandem mass spectrometry (LC-MS/MS); Piperaquine; Stable isotope labeled (SIL) internal standard; Solid-phase extraction;

Simultaneous high-performance liquid chromatographic determination of Cedrus deodara active constituents and their pharmacokinetic profile in mice by B.S. Sachin; M. Koul; A. Zutshi; S.K. Singh; A.K. Tikoo; M.K. Tikoo; A.K. Saxena; S.C. Sharma; R.K. Johri (237-241).
A specific and sensitive high-performance liquid chromatographic (HPLC) method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of three bioactive constituents of Cedrus deodara namely wikstromol, matairesinol and dibenzylbutyrolactol in mouse plasma. In solid-phase extraction (SPE) these constituents were successfully separated using a C18 column by isocratic elution using acetonitrile:water containing hexanesulphonic acid, 32:68 (v/v). The flow rate was set at 1 ml/min and detector wavelength at 225 nm. Good linearity (r 2  > 0.999) was observed over the studied range of 0.015–5.0 μg/ml for wikstromol and 0.030–5.0 μg/ml for matairesinol and dibenzylbutyrolactol. The CV values of intra-day precision for wikstromol, matairesinol and dibenzylbutyrolactol were in between 1.8–6.9, 1.7–4.9 and 1.6–4.2% and values of inter-day precision were in between 10.4–12.2, 9.7–11 and 10–11.2%, respectively. The extraction recoveries at low to high concentration were greater than 98, 83 and 87% for each analyte, respectively. The LOQ for wikstromol was 0.015 μg/ml and for both matairesinol and dibenzylbutyrolactol it was 0.030 μg/ml. The developed method was used to determine the pharmacokinetics of the three analytes in mice after intraperitoneal administration of CD-3.
Keywords: Cedrus deodara; Dibenzylbutyrolactol; Matairesinol; Pharmacokinetics; Wikstromol;

A simple and sensitive liquid chromatography–tandem mass spectrometry assay for the quantification of ertapenem in microdialysate by Sandrine Lefeuvre; Nicolas Venisse; Sandrine Marchand; Mickaël Bachelet; William Couet (242-245).
A new liquid chromatography assay with isocratic elution and tandem mass spectrometry detection (LC–MS/MS) using an electrospray ionization interface in the multiple reaction monitoring mode was developed and validated for ertapenem determination in microdialysate samples. Linearity was demonstrated between 10 ng mL−1 (lower limit of quantification, LLoQ) and 160 ng mL−1. The precision (CV%) and accuracy (bias%) in microdialysates at the LLoQ were respectively 2.2% and 17.3% within-day and 10.6% and 2.7% between-days. Ertapenem was stable for 1 month at −20 °C and −80 °C but unstable at +4 °C. This new LC–MS/MS assay is simple, rapid and more sensitive than previously described assays.
Keywords: LC–MS/MS; Ertapenem; Microdialysis; Pharmacokinetics;

A simple method using a one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) followed by high-performance liquid chromatography (HPLC) with negative-ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of cilnidipine in human plasma using benidipine as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 491.1 > 121.8 for cilnidipine and m/z 504.2 > 122.1 for IS, respectively. Analytes were chromatographed on a CN column by isocratic elution using 10 mM ammonium acetate buffer-methanol (30:70, v/v; adjusted with acetic acid to pH 5.0). Results were linear (r 2  = 0.99998) over the studied range (0.1–20 ng/ml) with a total LC–MS/MS analysis time per run of 3 min. The developed method was validated and successfully applied to a cilnidipine bioequivalence study in 24 healthy male volunteers.
Keywords: Cilnidipine; Liquid–liquid extraction; Liquid chromatography/tandem mass spectrometry; Human plasma; Bioequivalence;

The designer drug 2,5-dimethoxy-4-methyl-amphetamine (DOM, STP) is known to be extensively metabolized in various species. The current study showed that cytochrome P450 2D6 was the only isoenzyme involved in formation of the main metabolite hydroxy DOM. In addition, the authors’ systematic toxicological analysis (STA) procedure using full-scan GC–MS was suitable to prove an intake of a common drug users’ dose of DOM by detection of hydroxy DOM in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOM in human urine. However, DOM and/or other metabolites such as deamino-oxo-hydroxy DOM might be the target analyte in urine of CYP2D6 poor metabolizers.
Keywords: 2,5-Dimethoxy-4-methyl-amphetamine; DOM; STP; Designer drug; GC–MS; CYP2D6;

Simple and sensitive method for quantification of low tobramycin concentrations in human plasma using HPLC–MS/MS by Milly E. Attema-de Jonge; Judith M. Bekkers; Heleen M. Oudemans-van Straaten; Rolf W. Sparidans; Eric J.F. Franssen (257-262).
After oral administration of tobramycin, as part of selective decontamination of the digestive tract (SDD) in critically ill patients, absorption of tobramycin from the gut into the blood may take place. To quantify low concentrations of tobramycin in human plasma, we developed and validated a simple (sample pre-treatment consisting of protein precipitation with acetonitrile using 200 μl plasma), rapid (runtime 3 min using a Pathfinder MR reversed-phase column) and sensitive (concentration range of 0.05–1.0 mg/l using MS/MS detection) method.
Keywords: Tobramycin; Sisomicin; HPLC–MS/MS; Selective decontamination of the digestive tract;