Journal of Chromatography B (v.805, #1)

FM iii (iii).

NEWS Section (N1-N2).

A high-performance liquid chromatographic method is described for determination of lidocaine (2-(dietyloamino)-N-(2,6-dimetylofenylo) acetamid) and its metabolite, monoethylglycine xylidide (MEGX), in human serum containing various concentration of bilirubin. Lidocaine and its metabolite were extracted from human serum using dichloromethane. After separation of the layers and freezing at −32 °C, the organic layer was decanted and evaporated under a stream of nitrogen. The sample was dissolved in the mobile phase (12% acetonitrile in 15 mM potassium dihydrogen orthophosphate, pH 3.0), and after separation on a Supelcosil LC-8-DB column, the analytes were measured by ultraviolet detection at 205 nm. Trimethoprim (TMP) was used as the internal standard. The recovery of the examined analytes ranged from 95.7 to 97.9% for lidocaine and from 98.0 to 99.9% for MEGX. The lower limit of quantification (LLOQ) was established at 200 μg/l for lidocaine and at 10 μg/l for MEGX. The choice of suitable conditions for chromatographic separation of lidocaine and its metabolite MEGX allowed the elimination of the influence of endogenous bilirubin on the result of analysis.
Keywords: Lidocaine; Monoethylglycine xylidide; Bilirubin;

A rapid, sensitive and accurate liquid chromatographic–tandem mass spectrometric method is described for the determination of tamsulosin in dog plasma. Tamsulosin was extracted from plasma using a mixture of hexane–ethyl acetate (2:1, v/v) and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of methanol, water and formic acid (80:20:1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Atmospheric pressure chemical ionization (APCI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 409→m/z 228 and m/z 256→m/z 166.9 were used to quantify tamsulosin and the internal standard, respectively. The linearity was obtained over the concentration range of 0.1–50.0 ng/ml for tamsulosin and the lower limit of quantitation was 0.1 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 5.0 and 4.0% (relative standard deviation (R.S.D.)), respectively, and accuracy was within ±0.3% (relative error (R.E.)). This method was successfully applied to pharmacokinetic study of a tamsulosin formulation product after oral administration to beagle dogs.
Keywords: Tamsulosin;

Liquid chromatography–negative ion electrospray tandem mass spectrometry method for the quantification of tacrolimus in human plasma and its bioanalytical applications by N.V.S Ramakrishna; K.N Vishwottam; S Puran; S Manoj; M Santosh; S Wishu; M Koteshwara; J Chidambara; B Gopinadh; B Sumatha (13-20).
A simple, rapid, novel and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of tacrolimus (I) in human plasma, a narrow therapeutic index, potent macrolide immunosuppressive drug. The analyte and internal standard (tamsulosin (II)) were extracted by liquid–liquid extraction with t-butylmethylether using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra ODS column with a mobile phase of 99% methanol and 1% 10 mM ammonium acetate buffer. The deprotonate of analyte was quantitated in negative ionization by multiple reaction monitoring (MRM) with a mass spectrometer. The mass transitions m/z 802.5→560.3 and m/z 407.2→151.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.05–25 ng/ml for tacrolimus in human plasma. The lower limit of quantitation was 50 pg/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in comparative bioavailability studies. The tacrolimus plasma concentration profile could be obtained for pharmacokinetic study. The observed maximum plasma concentration (C max) of tacrolimus (5 mg oral dose) is 440 pg/ml, time to observed maximum plasma concentration (T max) is 2.5 h and elimination half-life (T 1/2) is 21 h.
Keywords: Tacrolimus;

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5′-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1–20 and 5–1000 ng/ml, are described. Following liquid–liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 μl) and part of the extract (1 μl) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25  m×0.2  mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 μm) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1–20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5–1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91–112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.
Keywords: Cyclooxygenase II inhibitor;

Sensitive and simple gas chromatographic–mass spectrometric determination for amphetamine in microdialysate and ultrafiltrate samples by Shan Xie; John Aspromonte; Andrea Balla; Henry Sershen; Daniel C. Javitt; Thomas B. Cooper (27-31).
A gas chromatographic–mass spectrometric (GC–MS) method is described for the measurement of amphetamine (AMP) using negative chemical ionization (NCI) mode. Without prior extraction AMP was derivatized with 2,3,4,5,6-pentafluorobenzoyl chloride (PFBC) and simultaneously extracted into toluene. The toluene extract was injected directly into GC–MS equipped with a HP-1 capillary column. The method is simple and more sensitive than most of the previously published methods. The limit of quantification of amphetamine is 25 pg (1.4 pg on column) with a very limited sample volume (25 μl). The within-day precision was from 1.7 to 5.1% and between-day precision was from 2.2 to 7.3%. The method has been used for the measurement of several thousand microdialysate and ultrafiltrate samples and proven reliable.
Keywords: Amphetamine;

Malondialdehyde (MDA) is considered a presumptive biomarker for lipid peroxidation in live organisms and cultured cells. The present study adapts an accurate and reproducible method to measure MDA by high-performance liquid chromatography (HPLC) as its 2,4-dinitrophenylhydrazone derivative in human hepatoma HepG2 cells in culture. Since MDA is assumed to increase in conditions of cellular oxidative stress, two compounds that induce pharmacological oxidative stress in cell cultures, hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BOOH), have been used in HepG2 cells. The results report a significant increase in the content of MDA derivative after treatment with 200 and 500 μM t-BOOH for 3 h, while H2O2 in doses up to 500 μM failed to evoke a similar response, indicating a stronger lipid peroxidation of t-BOOH to HepG2 cells than H2O2. Thus, MDA can be used as a reliable biomarker for cellular oxidative stress in human hepatoma HepG2.
Keywords: Derivatization, LC; Malondialdehyde; HepG2;

A new method, based on stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography–mass spectrometry (GC–MS) is described for the determination of trace amounts of bisphenol A (BPA) in river water, urine, plasma, and saliva samples. The derivatization conditions with acetic acid anhydride and the SBSE conditions such as sample volumes and extraction time are investigated. Then, the stir bar is subjected to TD followed by GC–MS. The detection limits of BPA in river water, urine, plasma, and saliva samples are 1–5, 20, 100, and 20 pg ml−1 (ppt), respectively. Calibration for BPA was shown to be linear with a correlation coefficient of >0.99. The average recoveries of BPA in all samples are higher than 95% (R.S.D. < 10%) with correction using an added surrogate standard, 13 C 12 -bisphenol A. This simple, accurate, sensitive, and selective analytical method may be applicable to the determination of trace amounts of BPA in liquid samples.
Keywords: Stir bar sorptive extraction; Derivatization, GC; Bisphenol A;

Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites by Antonia M. Calafat; A.Ryan Slakman; Manori J. Silva; Arnetra R. Herbert; Larry L. Needham (49-56).
While the demonstrated benefits associated with breastfeeding are well recognized, breast milk is one possible route of exposure to environmental chemicals, including phthalates, by breastfeeding infants. Because of the potential health impact of phthalates to nursing children, determining whether phthalates are present in breast milk is important. We developed a sensitive method for measuring 13 phthalate metabolites in breast milk using automated solid phase extraction (SPE) coupled to isotope dilution–high-performance liquid chromatography (HPLC)–negative ion electrospray ionization–tandem mass spectrometry. We used D4-phthalate diesters to unequivocally establish the presence in human breast milk of enzymes capable of hydrolyzing the ubiquitous phthalate diesters to their respective monoesters. The analytical method involves acid-denaturation of the enzymes after collection of the milk to avoid hydrolysis of contaminant phthalate diesters introduced during sampling, storage, and analysis. The method shows good reproducibility (average coefficient of variations range between 4 and 27%) and accuracy (spiked recoveries are ∼100%). The detection limits are in the low ng/ml range in 1 ml of breast milk. We detected several phthalate metabolites in pooled human breast milk samples, suggesting that phthalates can be incorporated into breast milk and transferred to the nursing child.
Keywords: Phthalates;

Dexamethasone (DXM) is a synthetic glucocorticoid that is authorized for therapeutic use in veterinary medicine. The European Community (EC) fixed a maximum residue limit (MRL) at 2 ng/g for liver, 0.75 ng/g for muscle and kidney tissues, and 0.3 ng/ml for milk, while its use as growth-promoter is completely banned. The purpose of this study was to develop and validate a simple and reliable method to determine DXM residues in bovine milk. Milk proteins were removed by the addition of concentrated trichloroacetic acid and paper filtration. Solid-phase extraction clean-up on a C18 reversed phase column was performed to obtain an extract suitable for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Chromatographic separation of DXM and the internal standard desoximetasone, was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.1% (v/v) acetic acid in water (mobile phase A) and acetonitrile (mobile phase B) as the mobile phases. They were identified using the MS/MS detection technique, and were subsequently quantified. The method has been validated according to the requirements of the EC at 0.15, 0.30 and 0.60 ng/ml (being half the MRL, the MRL and double the MRL levels fixed by the EC). Calibration graphs were prepared in the 0.15–5 ng/ml range and good linearity was achieved (r≥0.99 and goodness of fit ≤10%). A limit of quantification of 0.15 ng/ml, i.e. half the MRL, was obtained. The limit of detection was 41 pg/ml. The decision limit (CCα) and detection capability (CCβ) were 0.48 and 0.76 ng/ml, respectively. The within-day and between-day precisions, expressed as R.S.D. values, were all below the maximum allowed R.S.D. values calculated according to the Horwitz equation. The results for accuracy fell within the −50 to +20% range. Recovery was 56%. The method was used for the quantitative determination of DXM residues in milk after intravenous administration of DXM to lactating cows to determine its depletion kinetics.
Keywords: Dexamethasone;

We report here a quantitative method for the analysis of ABT-578 in human whole blood samples. Sample preparation was achieved by a semi-automated 96-well format liquid–liquid extraction (LLE) method. Aluminum/polypropylene heat seal foil was used to enclose each well of the 96-well plate for the liquid–liquid extraction. A liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method with pre-column regeneration was developed for the analysis of sample extracts. Selective reaction monitoring (SRM) of the mass transitions m/z 983–935 and m/z 931–883 was employed for the detection of ABT-578 and internal standard, respectively. The ammonium adduct ions [M + NH4]+ generated from electrospray ionization were monitored as the precursor ions. The assay was validated for a linear dynamic range of 0.20–200.75 ng/ml. The correlation coefficient (r) was between 0.9959 and 0.9971. The intra-assay CV (%) was between 1.9 and 13.5% and the inter-assay CV (%) was between 4.7 and 11.3%. The inter-assay mean accuracy was between 86.4 and 102.5% of the theoretical concentrations.
Keywords: ABT-578;

Measuring residual solvents in pharmaceutical samples using fast gas chromatography techniques by Marie-Josée Rocheleau; Mélanie Titley; Julie Bolduc (77-86).
Residual process solvents in pharmaceutical samples are monitored using gas chromatography (GC) with either flame ionization detection (FID) or mass spectrometry. Based on good manufacturing practices, measuring residual solvents is mandatory for the release testing of all active pharmaceutical ingredients and is routinely performed on samples of process intermediates. General GC methods have been developed to monitor solvents routinely used in the drug synthesis process. It is now possible to take advantage of GC equipment with faster temperature ramping capabilities, in combination with shorter capillary GC columns, to achieve a considerable gain in efficiency and a reduction in analysis turnaround time. In this paper, the development and implementation of fast GC methods for residual solvents testing will be discussed. Continued efforts to improve the efficiency of gas chromatography using existing technologies such as, the ThermoOrion Flash GC will also be discussed.
Keywords: Residual solvents; Fast gas chromatography; Pharmaceutical samples;

Determination of perhexiline and hydroxyperhexiline in plasma by liquid chromatography–mass spectrometry by O. Beck; N. Stephanson; R.G. Morris; B.C. Sallustio; P. Hjemdahl (87-91).
A method for the quantitative determination of perhexiline and its main hydroxylated metabolites in human plasma, based on liquid chromatography–mass spectrometry (LC–MS), was developed. The method used protein precipitation with acetonitrile followed by dilution with water and subsequent direct injection of the extract into the LC–MS system. Hexadiline was used as internal standard and the intra-assay coefficients of variation were ≤5% for perhexiline and cis-hydroxyperhexiline over the target concentration range in patients. The lower limits of quantification were 0.005 mg/l for perhexiline and 0.015 mg/l for cis-hydroxyperhexiline, and the measuring ranges were from 0.05 to 3.0 and from 0.2 to 6.0 mg/l, respectively. The method was compared with an established HPLC method with fluorescence detection and the correlation between the methods was close to 1 for both compounds. The predominant form of hydroxyperhexiline in 87% of the patient samples was found to be one of the diastereomeric pairs of cis-hydroxyperhexiline. In patients not forming this metabolite, trans-hydroxyperhexiline could be detected. We conclude that the present LC–MS method is suitable for use in a clinical routine laboratory.
Keywords: Perhexiline; Hydroxyperhexiline;

6-Aminobutylphthalide (ABP) is a new drug candidate which is currently being developed for the treatment of cerebral ischemia. The pharmacokinetics and metabolism of ABP were studied using in situ microdialysis sampling in the brains of awake freely-moving rats. Two LC-MS/MS methods were used for the quantitative and qualitative analysis of microdialysate. For comparison and confirmation, brain tissue samples were also analyzed by LC-MS/MS and GC/MS. The results described provide more authentic information in pharmacokinetics and metabolism at the site of action by using the coupling of microdialysis to LC-MS/MS technique than the traditional sampling methods.
Keywords: 6-Aminobutylphthalide;

A simple and reproducible reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method using isocratic elution with UV absorbance detection is presented for the simultaneous quantitation of mycophenolic acid (MPA) and MPA-glucuronide (MPAG) in human plasma and urine. The sample preparation procedures involved simple protein precipitation for plasma and 10-fold dilution for urine. Each analytical run was completed within 15 min, with MPAG and MPA being eluted at 3.8 and 11.4 min, respectively. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.
Keywords: Mycophenolic acid; Mycophenolic acid glucuronide;

We devised a sensitive and simple method for simultaneous determination of formate and acetate in whole blood and urine from humans using gas chromatography–mass spectrometry. Formate and acetate were alkylated with pentafluorobenzyl bromide in the mixture of acetone and phosphate buffer (pH 6.8). The derivatives obtained were analyzed using gas chromatography–mass spectrometry in positive-ion electron ionization (EI) mode. The lower limit of detection for both compounds was 0.02 mM. The calibration curves for formate and acetate were linear over the concentration range from 0.05 to 5.0 mM. Accuracy and precision of the method were evaluated and the coefficients of variation were within 10%. With use of this method, levels of formate and acetate in whole blood can be determined in forensic cases.
Keywords: Formate; Acetate;

By selecting the different combination schemes, a simple, fast and highly efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese cobra is reported in this paper. This purification process consists of a two-step chromatographic separation on DEAE-Sepharose F.F. anion-exchange medium followed by a Sephadex G-50 gel filtration. On reducing and non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the nerve growth factor obtained with this process proved to be homogeneous and its molecular weight was separately estimated to be approximately 14.5 and 29.0 kD, which was consistent with that reported in literature; and on high performance size-exclusion chromatography and reversed-phase chromatography, its purity was about 99%. The yield of this purification method was 0.51% and the nerve growth factor obtained had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. The optimum concentration of nerve growth factor was 5–100 ng/ml and the minimal concentration eliciting neurite outgrowth from chick embryonic dorsal root ganglia was 5.0 ng/ml.
Keywords: Chinese cobra venom; Nerve growth factor;

Globotriaosylceramide isoform profiles in human plasma by liquid chromatography–tandem mass spectrometry by Bryant C Nelson; Thomas Roddy; Shaparak Araghi; Dennis Wilkens; John J Thomas; Kate Zhang; Crystal C.-C Sung; Susan M Richards (127-134).
Globotriaosylceramide (GL3) is a heterogeneous glycosphingolipid that is elevated in the blood plasma of patients diagnosed with Fabry disease. GL3 consists of numerous isoforms, some of which are distinctly specific to human plasma. An electrospray-ionization LC/MS/MS method has been developed that has the capacity to monitor the GL3 isoform profiles in plasma extracts. Total GL3 is extracted from human plasma via chloroform/methanol liquid–liquid extraction, purified by C18 solid-phase extraction and analyzed by multiple reaction monitoring LC/MS/MS. The relative responses of eight selected isoforms are calculated on the basis of the total GL3 response and the isoform responses are subsequently utilized to construct isoform profile plots.
Keywords: Globotriaosylceramide; Lipids;

Determination of SU5416, a novel angiogenesis inhibitor, in human plasma by liquid chromatography by Tanyifor M Tohnya; Sonia Kim; Howard A Fine; Lara Dunn; William D Figg; Alex Sparreboom (135-140).
A high-performance liquid chromatographic (HPLC) assay with UV detection has been developed for the quantitative determination of the antiangiogenic agent SU5416 in human plasma. Sample pretreatment involved a single protein-precipitation step with acetonitrile containing the internal standard, chrysin. Separation of the compounds of interest was achieved on a column packed with HP Zorbax C8 material (5 μm particle size; length: 150 mm; i.d.: 4.6 mm) using a dual solvent system of 0.01 M aqueous ammonium acetate and acetonitrile delivered as a nonlinear gradient at a flow-rate of 1.00 ml/min. Simultaneous UV detection was performed at 440 nm (SU5416) and 268 nm (chrysin). The calibration graph was fit to log-transformed response-concentration data over a range of 10–5000 ng/ml. Values for accuracy and precision, obtained from six quality controls analyzed on different days in replicates of 3 or 6, ranged 92.9–109 and 0.8–6.2%, respectively. The developed method was successfully applied to study the pharmacokinetics of SU5416 in a cancer patient receiving the drug as a 1 h infusion.
Keywords: SU5416; Angiogenesis inhibitor;

Determination of human plasma levels of levo-α-acetylmethadol and its metabolites by gas chromatography–mass spectrometry by C.B. Eap; G. Bouchoux; N. Scherbaum; M. Gastpar; K. Powell Golay; P. Baumann (141-146).
A gas chromatography–mass spectrometry (GC–MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-α-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5–200 ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4 ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM’s blood levels, clinical efficacy and/or cardiotoxicity
Keywords: Levo-α-acetylmethadol;

Improved assay for determination of busulfan by liquid chromatography using postcolumn photolysis by Andreas Jenke; Ulf Renner; Ulrich S Schuler; Sylvia Wauer; Traugott Leopold; Eberhard Schleyer; Gerhard Ehninger (147-153).
A highly sensitive and time-reduced HPLC assay for the quantitative analysis of busulfan in plasma and aqueous samples is described. The assay is based on a precolumn derivatization of busulfan to 1,4-diiodobutane and UV-detection of iodide ions generated by a postcolumn photochemical dissociation of the derivative. The extraction and derivatization were carried out in a one-pot reaction without any solid phase extraction and is therefore suitable for high throughput analysis. Quantification was performed by using 1,5-pentanediol-bis-(methanesulfonate), a homologue of busulfan, as an internal standard. Linearity was demonstrated for concentrations from 50 to 10,000 ng/ml. The limit of detection was found at 10 ng/ml. Precision is indicated by an intra-day variety of 2.81% and by an inter-day variety of 6.61% for aqueous samples, 2.93 and 5.76% for plasma samples, respectively. The recovery of busulfan in plasma was more than 95%. No coelution with metabolites of busulfan or other drugs used in cancer therapy was found. The method was generated for measurements of busulfan in aqueous or plasma samples and applied in therapeutic drug monitoring of busulfan.
Keywords: Busulfan;

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000–6% (w/w) dextran T500 containing 10 mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0 ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500 mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.
Keywords: Streptococcus mutans; Glucosyltransferase;

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction by Manori J. Silva; A.Ryan Slakman; John A. Reidy; James L. Preau; Arnetra R. Herbert; Ella Samandar; Larry L. Needham; Antonia M. Calafat (161-167).
We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.
Keywords: Phthalate metabolites;

Liquid chromatographic method for the determination of rizatriptan in human plasma by Jun Chen; Xinguo Jiang; Wenming Jiang; Ni Mei; Xiaoling Gao; Qizhi Zhang (169-173).
A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the determination of rizatriptan in human plasma. Following a single-step liquid–liquid extraction with methyl tertiarybutyl ether, the analytes were separated using a mobile phase consisting of 0.05% (v/v) triethylamine in water (adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v). Fluorescence detection was performed at an excitation wavelength of 225 nm and an emission wavelength of 360 nm. The linearity for rizatriptan was within the concentration range of 0.5–50 ng/ml. The intra- and inter-day precisions of the method were not more than 8.0%. The lower limit of quantification (LLOQ) was 0.5 ng/ml for rizatriptan. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.
Keywords: Rizatriptan;

This study describes a new simultaneous determination of haloperidol and bromperidol and their reduced metabolites by modification of automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1 ml of plasma using chloroform–hexane (30:70 (v/v)), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C18 analytical column for separation. The mobile phases consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (60%) and acetonitrile (54:1:45 (v/v)) and was delivered at a flow-rate of 0.6 ml/min. The peak was detected using a UV detector set at 215 nm. The method was validated for the concentration range 1–100 ng/ml, and good linearity (r>0.999) was confirmed. Intra-day coefficient variations (CVs) for haloperidol, reduced haloperidol, bromperidol and reduced bromperidol were less than 2.5, 3.1, 2.4 and 2.5%, respectively. Inter-day CVs for corresponding compounds were 3.9, 5.1, 2.6 and 4.4%, respectively. Relative errors ranged from −5 to 10% and mean recoveries were 96–100%. The limit of quantification was 1.0 ng/m for each compound. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring, particularly in patients receiving both haloperidol and bromperidol.
Keywords: Haloperidol; Bromperidol;

A liquid chromatography–tandem mass spectrometric (LC–MS–MS) method with a rapid and simple sample preparation was developed and validated for the determination of Tirofiban in biological fluids. Tirofiban in serum samples was extracted and cleaned up by using an automated solid phase extraction method. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode (MRM). A good linear response over the range of 2–200 ng/ml was demonstrated. The accuracy for Tirofiban ranged from 94.8 to 110.8% within-day and from 103.0 to 104.7% between-day. The lower limit of quantification was 2 ng/ml. This method is suitable for pharmacokinetic studies.
Keywords: Tirofiban;