Journal of Chromatography B (v.830, #2)

Quantification of six herbicide metabolites in human urine by Jessica Norrgran; Roberto Bravo; Amanda M. Bishop; Paula Restrepo; Ralph D. Whitehead; Larry L. Needham; Dana B. Barr (185-195).
We developed a sensitive, selective and precise method for measuring herbicide metabolites in human urine. Our method uses automated liquid delivery of internal standards and acetate buffer and a mixed polarity polymeric phase solid phase extraction of a 2 mL urine sample. The concentrated eluate is analyzed using high-performance liquid chromatography–tandem mass spectrometry. Isotope dilution calibration is used for quantification of all analytes. The limits of detection of our method range from 0.036 to 0.075 ng/mL. The within- and between-day variation in pooled quality control samples range from 2.5 to 9.0% and from 3.2 to 16%, respectively, for all analytes at concentrations ranging from 0.6 to 12 ng/mL. Precision was similar with samples fortified with 0.1 and 0.25 ng/mL that were analyzed in each run. We validated our selective method against a less selective method used previously in our laboratory by analyzing human specimens using both methods. The methods produced results that were in agreement, with no significant bias observed.
Keywords: Herbicide; Urine; Biomonitoring; HPLC–MS/MS; Chloroacetanilide; Alachlor; Acetochlor; Metolachlor; Triazine; Atrazine; 2,4-D;

A novel, quantitative assay for homocarnosine in cerebrospinal fluid using stable-isotope dilution liquid chromatography–tandem mass spectrometry by Erwin E.W. Jansen; K. Michael Gibson; Yosuke Shigematsu; Cornelis Jakobs; Nanda M. Verhoeven (196-200).
We describe a rapid and sensitive method for the quantification of homocarnosine in physiological fluids, with particular emphasis on cerebrospinal fluid (CSF). Homocarnosine was quantified as the butyl derivative, with 2H2-l-homocarnosine as internal standard. Following deproteinization of CSF samples, supernatants were evaporated to dryness and derivatized with 10% 6 M HCl in butanol. Samples were chromatographed on a C18 column and detected by liquid chromatography–tandem mass spectrometry (LC–MS/MS) operating in the multiple reaction monitoring mode. The intra- and inter-assay variations were 4.6 and 10.9%, respectively. Mean recovery of homocarnosine at two concentrations was 105%. The limit of detection in CSF approximated 20 nmol/L. CSF homocarnosine is age dependent and ranges from <0.02 to 10 μmol/L. Our method is applicable to the analysis of CSF derived from patients with heritable defects in the GABA pathway, patients with homocarnosinosis or serum carnosinase deficiency, and should be applicable to other model systems in order to further explore the biological role and significance of homocarnosine in mammalian systems.
Keywords: Homocarnosine; GABA; Tandem mass spectrometry; Succinic semialdehyde dehydrogenase deficiency;

A new, simple and rapid high-performance liquid chromatography (HPLC) method with UV detection has been developed for the determination of apovincaminic acid in human plasma. Apovincaminic acid and internal standard were isolated from plasma samples by solid-phase extraction with OASIS® HLB cartridges. The chromatographic separation was accomplished on a reversed-phase C18 column and UV detection was set at 311 nm. The calibration curves were linear in the concentration range of 2.4–240.0 ng/ml, and the limits of quantification was 2.4 ng/ml. The precision and accuracy ranged from 0.84 to 8.54% and 91.5 to 108.3%, respectively. The developed method was subsequently applied to study the pharmacokinetics of apovincaminic acid in a group of 20 human subjects at a single oral dose of 10 mg of vinpocetine tablet.
Keywords: Apovincaminic acid; High-performance liquid chromatography (HPLC);

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma by Mohammad-Reza Rouini; Yalda Hosseinzadeh Ardakani; Faezeh Soltani; Hassan Y. Aboul-Enein; Alireza Foroumadi (207-211).
Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a ChromolithTM Performance RP-18e 50 mm × 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (λ ex  = 200 nm/λ em  = 301 nm) was used. The calibration curves were linear (r 2  > 0.997) in the concentration range of 2.5–500 ng/ml, 1.25–500 ng/ml and 5–500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5–9.7%, 2.5–9.9% and 5.9–11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100 mg single oral dose of tramadol to healthy volunteers.
Keywords: Tramadol; O-Desmethyltramadol; N-Desmethyltramadol; Plasma; Monolithic; Pharmacokinetics;

A new extraction method has been developed for the extraction of prostaglandin E2 (PGE2) from human plasma of patients suffering chronic inflammatory disorders. The extraction solvents were optimised systematically and simultaneously by using a central composite design. The optimised method involves precipitation of the protein fraction, centrifugation, evaporation and dissolution of the supernatant in the mobile phase, screening to confirm the presence of the analyte, and quantification of the positive samples by liquid chromatography tandem ion-trap mass spectrometry. Tandem mass spectrometry in negative mode was performed by isolating and fragmenting the ion [PGE2-H] signal m/z 351. Identification and quantification was carried out by extracting the ion fragment chromatograms at 333, 315 and 271 m/z. The quantitative determination was linear for the low nanogram (1–50 ng/ml) and upper picogram (400–1000 pg/ml) range studied, using 15 and 0.5 ng/ml of internal standard, respectively. The lower limit of detection was 2.5 pg for an injection volume of 25 μl. The optimised extraction method showed high reproducibility (coefficients of variation < 4%) and recovery values, estimated from standard addition experiments, ranging from 96 to 98%.
Keywords: Prostaglandin E2; LCMS; Experimental design; Central composite design; Ion-trap mass spectrometry; Plasma extraction method;

Determination of free ertapenem in plasma and bronchoalveolar lavage by high-performance liquid chromatography with ultraviolet detection by Jean-Baptiste Gordien; Emmanuel Boselli; Catherine Fleureau; Bernard Allaouchiche; Gérard Janvier; Olivier Lalaude; Marie-Claude Saux; Dominique Breilh (218-223).
A sensitive assay for the determination of unbound ertapenem in human plasma and bronchoalveolar lavage (BAL) was developed using ultrafiltration of plasma and BAL samples. A rapid HPLC method was used with ultraviolet detection set at a wavelength of 305 nm and a separation on a Prontosil AQ C18 column, with imipenem used as internal standard. This assay was linear over the concentration range of 0.5–100 μg/mL and 0.25–50 μg/mL in plasma and BAL, respectively. Limits of detection and quantitation were respectively 0.05 and 0.25 μg/mL. Validation data for accuracy and precision were CV < 2.48 and 8.25%, accuracy in the range 98.1–104.2% and 102.2–108.4%, respectively, for intra and inter-day.
Keywords: Free ertapenem in plasma and BAL; Ultrafiltration; HPLC UV detection; Pharmacokinetics;

A capillary electrophoresis with electrochemical detection (CE-ED) method was developed for the simultaneous determination of four major flavonoid markers (synephrine, naringin, hesperidin and naringenin) in Frucus aurantii of different geographical origin. Operated in a wall-jet configuration, a 300 μm diameter carbon disc electrode was used as the working electrode, which exhibits a good response at +0.85 V (versus saturated calomel electrode) for the analytes. Under the optimum conditions, the analytes were baseline separated within 20 min in a 80 mmol/L borax buffer (pH 8.45). The intra-day relative standard deviations (R.S.D.) and inter-day R.S.D.s were based on the analysis of the standard solution on the same day and on the following 6 consecutive days. The intra-day R.S.D.s ranged from 0.8% (naringin) to 3.6% (hesperidin). The inter-day R.S.D.s ranged from 1.2% (hesperidin) to 4.6% (naringenin). Calibration curves were linear in ranges between 0.05 and 1000 μg/mL for the markers. Limits of detection ranged from a low of 1 × 10−8  g/mL (hesperidin) to a high of 5 × 10−7  g/mL (naringin). The method was successfully used in the analysis of F. aurantii of different geographical origin with relatively simple extraction procedures, and the assay results were satisfactory.
Keywords: Frucus aurantii; Capillary electrophoresis; Electrochemical detection; Flavonoids;

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4′-hydroxydiclofenac in rat serum by Lata Kaphalia; Bhupendra S. Kaphalia; Santosh Kumar; Mary F. Kanz; Mary Treinen-Moslen (231-237).
A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4′-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4′-hydroxydiclofenac were 0.0225 and 0.0112 μg/ml, respectively. Average extraction efficiencies of diclofenac, 4′-hydroxydiclofenac, and the internal standard were ≥76%. The method was applied to serum collected at 3 h after rats were treated with an experimentally useful dosage range of 3, 10 and 50 mg/kg diclofenac. Recovery (as a percentage of dose) for the 4′-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.
Keywords: Diclofenac; 4-Hydroxydiclofenac; Metabolism; Cytochrome P450 3A4;

Protein–drug interactions of seven common pharmaceuticals were studied using solid-phase microextraction (SPME). SPME can be used in such investigations on the condition that no analyte depletion occurs. In multi-compartment systems (e.g. a proteinaceous matrix) only the free portion of the analyte is able to partition into the SPME fiber. In addition if no sample depletion occurs, the bound drug-free drug equilibria are not disturbed. In the present study seven pharmaceuticals (quinine, quinidine, naproxen, ciprofloxacin, haloperidol, paclitaxel and nortriptyline) were assayed by SPME. For quantitative purposes SPME was validated first in the absence of proteins. Calibration curves were constructed for each drug by HPLC-fluorescence and HPLC-UV analysis. SPME was combined to HPLC off-line, desorption occurring in HPLC inserts filled with 200 μL methanol. Binding of each drug to human serum albumin was studied independently. Experimental results were in agreement with literature data and ultrafiltration experiments, indicating the feasibility of the method for such bioanalytical purposes.
Keywords: Protein binding; Quinine; Quinidine; Naproxen; Ciprofloxacin; Haloperidol; Paclitaxel; Nortriptyline; Solid-phase microextraction; SPME;

Determination of celecoxib in human plasma and breast milk by high-performance liquid chromatographic assay by Mei Zhang; Grant A. Moore; Sharon J. Gardiner; Evan J. Begg (245-248).
A rapid and simple HPLC assay was developed for the determination of celecoxib in human plasma and breast milk. After proteins were precipitated with acetonitrile, celecoxib was resolved on a C18 column and detected by UV detection at 254 nm. Standard curves were linear over the concentration range 10–2000 μg/L (r 2  > 0.99). Bias was ≤±15% from 20 to 2000 μg/L in both matrices, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 10 μg/L.
Keywords: Celecoxib; Plasma; Milk; HPLC;

Simultaneous analysis of human plasma catecholamines by high-performance liquid chromatography with a reversed-phase triacontylsilyl silica column by Maiko Machida; Ayumi Sakaguchi; Satoshi Kamada; Tetsuya Fujimoto; Shigeru Takechi; Shigeo Kakinoki; Akikazu Nomura (249-254).
The clinical importance of simultaneous analysis of 3,4-dihydroxyphenylglycol with other human plasma catecholamines has been investigated to better understand the sympathetic nervous system. However, previous reports have had analytical difficulties with both resolution and extraction. The current study uses a reversed-phase triacontylsilyl silica (C30) column under the mobile phase condition without ion-pair reagents to separate catecholamines and their metabolites, with above 91% recoveries for intra-assay, above 85% for inter-assay, and less than 10% (n  = 5) coefficient of variation. Lower detection limits (S/N = 4) and quantification limits (S/N = 6) were 40 and 100 pg/mL for norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylalanine, 10 and 20 pg/mL for epinephrine, 10 and 40 pg/mL for dopamine. Linear ranges were from 40 to 5000 pg/mL for norepinephrine and 3,4-dihydroxyphenylalanine, from 100 to 5000 pg/mL for 3,4-dihydroxyphenylglycol, and from 10 to 2000 pg/mL for epinephrine and dopamine. The C30 column may prove clinically useful, as it provides a convenient and simultaneous method of evaluation of human plasma catecholamines.
Keywords: Norepinephrine; 3,4-Dihydroxyphenylglycol; Triacontylsilyl silica (C30); Solid-phase extraction;

O 6-Methyl-2′-deoxyguanosine (O 6-mdGuo), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), and 1,N 6-etheno-2′-deoxyadenosine (ɛdAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O 6-[2H3]mdGuo was synthesized and used as internal standard. The limits of quantification for O 6-mdGuo, 8-oxodGuo, and ɛdAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24–125 pmol/ml, 0.98–125 pmol/ml, and 0.49–62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O 6-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for ɛdAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O 6-mdGuo was above the limit of detection (37 adducts per 109 normal nucleosides) but could not be quantified. 8-oxodGuo and ɛdAdo showed background levels of 500 and 130 adducts per 109 normal nucleosides, respectively. DNA analyzed 1 h after treatment of rats with dimethylnitrosamine by oral gavage of 50 μg/kg b.wt. did not affect the levels of 8-oxodGuo and ɛdAdo but resulted in 200 O 6-mdGuo adducts per 109 normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.
Keywords: DNA adducts; Oxidative stress; Background DNA damage; Methylation; Oxidation; Lipid peroxidation; Rat liver; Mass spectrometry;

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography–protein digestion–liquid chromatography–mass spectrometry by Johannes S. Hoos; Hilke Sudergat; Jens-Peter Hoelck; Mark Stahl; Jon S.B. de Vlieger; Wilfried M.A. Niessen; Henk Lingeman; Hubertus Irth (262-269).
A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography–mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 μl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.
Keywords: Immunoaffinity; On-line analysis; Trypsin digestion; Protein; Peptide; Electrospray; Quantification; LC–MS;

Caco-2 cells are frequently used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential. Bi-directional permeability studies performed on Caco-2 cells followed by analysis by HPLC-UV or LC–MS method constitutes the “method of choice” for the functional assessment of efflux characteristics of a test compound. A high throughput LC–MS/MS method has been developed using on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection to analyze multiple compounds present in Hanks balanced salt solution in a single analytical run. All standard curves (P-gp substrates: quinidine, etoposide, rhodamine 123, dexamethasone, and verapamil and non-substrates: metoprolol, sulfasalazine, propranolol, nadolol, and furosemide) were prepared in a cassette mode (ten-in-one) while Caco-2 cell incubations were performed both in discreet mode and in cassette mode. The standard curve range for most compounds was 10–2500 nM with regression coefficients (R 2) greater than 0.99 for all compounds. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio greater than 1 for the P-gp substrates studied in the Caco-2 cell model. The use of cassette mode analysis through selected reaction monitoring mass spectrometry presents an attractive option to increase the throughput, sensitivity, selectivity, and efficiency of the model over discreet mode UV detection.
Keywords: Turbulent flow chromatography; Tandem mass spectrometry; Caco-2; LC–MS/MS analysis; On-line extraction;

Direct determination of the ratio of tetrahydrocortisol + allo-tetrahydrocortisol to tetrahydrocortisone in urine by LC–MS–MS by Andrea Raffaelli; Alessandro Saba; Edda Vignali; Claudio Marcocci; Piero Salvadori (278-285).
The 11β-hydroxysteroid dehydrogenase (11β-HSD) is responsible for the interconversion of both the hormonally inactive cortisone and the active cortisol. This enzyme activity, which has implications in the pathogenesis of numerous diseases, is reflected in the ratio of tetrahydrometabolites of cortisol (allo-tetrahydrocortisol and tetrahydrocortisol) to those of cortisone (tetrahydrocortisone). Several methods have been proposed in the literature to determine such a ratio in urine. Most of them require tedious and extensive extraction and derivatization steps and make use of gas-chromatographic techniques, including gas chromatography coupled to mass spectrometry (GC–MS). We present here an alternative approach for the direct determination of such a ratio in urine by using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS–MS), based on a minimal sample treatment. Actually, the limit of detections (LODs) for pure standards in water permitted a simple dilution of the urine samples prior to the analysis, hence, an accurate optimization of the high performance liquid chromatography (HPLC) separation was needed in order to get rid of the severe influence of the urine matrix on the ionization efficiency. Besides, the nature of some interfering species was deeply investigated, as well as the suitability of some commercial deuterated steroids as internal standards. All these led to the final method, which was based on a HPLC separation on a C8 column and a ternary gradient water/methanol/acetonitrile. In parallel, an appropriate sample preparation was set up, which consisted of an enzymatic hydrolysis of the conjugated species and a followed 1:20 dilution. Preliminary measurements on real urine samples were performed as well.
Keywords: Steroids; Metabolism; Mass spectrometry; Electrospray ionization;

Polyethyleneglycol molecular mass and polydispersivity effect on protein partitioning in aqueous two-phase systems by Guillermo Picó; Diana Romanini; Bibiana Nerli; Beatriz Farruggia (286-292).
The partitioning of model proteins (bovine serum albumin, ovalbumin, trypsin and lysozyme) was assayed in aqueous two-phase systems formed by a salt (potassium phosphate, sodium sulfate and ammonium sulfate) and a mixture of two polyethyleneglycols of different molecular mass. The ratio between the PEG masses in the mixtures was changed in order to obtain different polymer average molecular mass. The effect of polymer molecular mass and polydispersivity on the protein partition coefficient was studied. The relationship between the logarithm of the protein partition coefficient and the average molecular mass of the phase-forming polymer was found to depend on the polyethyleneglycol molecular mass, the salt type in the bottom phase and the molecular weight of the partitioned protein. The polymer polydispersivity proved to be a very useful tool to increase the separation between two proteins having similar isoelectrical point.
Keywords: Polyethyleneglycol; Polydispersivity; Partitioning; Aqueous two-phase systems;

A simple experimental approach for studying and identifying the relative matrix effect (for example “plasma-to-plasma” and/or “urine-to-urine”) in quantitative analyses by HPLC–MS/MS is described. Using as a database a large number of examples of methods developed in recent years in our laboratories, the relationship between the precision of standard line slopes constructed in five different lots of a biofluid (for example plasma) and the reliability of determination of concentration of an analyte in a particular plasma lot (or subject) was examined. In addition, the precision of standard line slopes was compared when stable isotope-labeled analytes versus analogs were used as internal standards (IS). Also, in some cases, a direct comparison of standard line slopes was made when different HPLC–MS interfaces (APCI versus ESI) were used for the assay of the same compound, using the same IS and the same sample preparation and chromatographic separation conditions. In selected cases, the precision of standard line slopes in five different lots of a biofluid was compared with precision values determined five times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 3–4% for the method to be considered reliable and free from the relative matrix effect liability. Based on the results presented, in order to assess the relative matrix effect in bioanalytical methods, it is recommended to perform assay precision and accuracy determination in five different lots of a biofluid, instead of repeat (n  = 5) analysis in the same, single biofluid lot, calculate standard line slopes and precision of these slopes, and to use <3–4% slope precision value as a guide for method applicability to support clinical studies. It was also demonstrated that when stable isotope-labeled analytes were used as internal standards, the precision of standard line slopes in five different lots of a biofluid was ≤2.4% irrespective of the HPLC–MS interface utilized. This clearly indicated that, in all cases studied, the use of stable isotope-labeled IS eliminated relative matrix effect. Also, the utilization of the APCI interface instead of ESI led to the elimination of the relative matrix effect in all cases studied. When the precision of standard line slope values exceeds the 3–4% limit, the method may require improvements (a more efficient chromatography, a more selective extraction, a stable isotope-labeled IS instead of an analog as an IS, and/or a change in the HPLC–MS interface) to eliminate the relative matrix effect and to improve assay selectivity.
Keywords: Bioanalysis; Relative matrix effect; Standard line slopes; HPLC; Tandem mass spectroscopy;

Column-switching high-performance liquid chromatographic method for the determination of zaltoprofen in rat plasma by Sun Ok Choi; So Young Um; Sung Hee Jung; Seo Jeong Jung; Joo Il Kim; Hwa Jeong Lee; Soo Youn Chung (301-305).
A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10 mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile–10 mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40–5000 ng mL−1. This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen.
Keywords: Zaltoprofen; Column switching; High-performance liquid chromatography (HPLC); Rat plasma; Direct injection;

A simple and selective micellar electrokinetic chromatography (MEKC) is described for determination of indomethacin in plasma. Plasma proteins are precipitated by acetonitrile. An aliquot of supernatant was evaporated and reconstituted with Tris buffer for MEKC analysis. The separation of indomethacin was performed at 25 °C using a background electrolyte consisting of Tris buffer (30 mM; pH 8.0) with 100 mM sodium octanesulfonate (SOS) as an anionic surfactant. Under this condition, a good separation with high efficiency and short analysis time is achieved. Several parameters affecting the separation of indomethacin were studied, including pH and concentrations of the Tris buffer and SOS. The linear range of the method for the determination of indomethacin was over 0.3–10.0 μg/mL; the detection limit (signal-to-noise ratio = 3; injection 0.5 psi 5 s) was 0.1 μg/mL. The proposed method for determination of indomethacin in premature infants with patent ducts arteriosus has been demonstrated.
Keywords: Indomethacin; Micellar electrokinetic chromatography; Plasma; Patent ducts arteriosus;

A sensitive, simple and fast liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 1-(4-chlorophenyl)biguanide (4CPB), was developed and validated over a concentration range of 1–2000 ng/mL using only 50 μL of blood or plasma. After a simple solvent precipitation procedure, the supernatant was analysed directly by HPLC-MS/MS. Separation was achieved using an ethyl-linked phenyl reverse phase column with polar endcapping with an acetonitrile–water–formic acid gradient. Mass spectrometry was performed using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The elution of PG (254.07 → 169.99), CG (252.12 → 195.02) and 4CPB (212.06 → 153.06) was monitored using selected reaction monitoring. The three compounds and the internal standard (chloroproguanil) were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in blood and plasma. The limit of quantification of PG and CG was 1 ng/mL and 5 ng/mL for 4CPB in rat blood and plasma. The extraction efficiency of PG, CG and 4CPB from rat blood and plasma was higher than 73%. The intra- and inter-assay variability of PG, CG and 4CPB were within 12% and the accuracy within ±5%. This new assay offers higher sensitivity and a much shorter run time over earlier methods.
Keywords: Proguanil; Cycloguanil; Antimalarial; LC–MS; Blood and plasma assay;

A sensitive and simple method based on solid-phase extraction (SPE) and HPLC with fluorescence detection for the determination of bisphenol A (BPA) and 4-nonylphenol (4-NP) in rat serum, liver and testis tissues has been developed. The chromatographic conditions consisted of a C18 column and mobile phase composition of acetonitrile and water with flow rate of 1.0 ml/min. The fluorescence detection was performed at excitation and emission wavelengths of 227 nm and 313 nm, respectively. Under these conditions, BPA and 4-NP were well separated and showed good linearities in the ranges of 0.01–50.0 μg/ml for BPA and 0.15–150.0 μg/ml for 4-NP with correlation coefficients greater than 0.999. The detection limits of serum and tissue samples were 2.8 ng/ml and 1.4 ng/g for BPA and 5.6 ng/ml and 2.8 ng/g for 4-NP at a signal-to-noise ratio (S/N) of 3. The intra-assay and the inter-assay precisions were better than 11.4%. Recoveries of BPA and 4-NP were 78.6–95.0% and 80.2–93.4%, respectively. The proposed method was applied to a toxicokinetic study of BPA and 4-NP including individual and combined oral administration to rats. The results showed that 4-NP remarkably altered the toxicokinetic parameters of BPA in testis, while parameters of BPA were not obviously altered in serum and liver under the experimental conditions investigated. On the other hand, there was no significant difference in the toxicokinetics of 4-NP when administered with BPA.
Keywords: HPLC; Environmental estrogens; Bisphenol A; 4-Nonylphenol; Solid-phase extraction; Rat serum and tissue; Toxicokinetics;

An ammonium-adduct based liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous determination of three isomeric metabolites of tacrolimus (FK506), 13-O-demethylated (M1), 31-O-demethylated (M2) and 15-O-demethylated (M3) tacrolimus in human whole blood and plasma. These metabolites and the internal standards were extracted from biological matrix by methylbutyl ether (MTBE). Separation was achieved on a Genesis C18 column with a gradient mobile phase elution. Ammonium-adduct ions formed by a Turbo Ionspray in positive ion mode were used to detect each analyte and internal standard. The MS/MS detection was by monitoring the fragmentation of 807.5 → 772.4 (m/z) for M1, 807.5 → 754.5 (m/z) for both M2 and M3, 795.5 → 760.5 (m/z) for IS1 (FR298701) and 961.5 → 908.5 (m/z) for IS2 (FR290198) on a triple quadrupole mass spectrometer (Sciex API 3000). The retention times were approximately 4.1 min for M1, 6.8 min for M2, 6.0 min for M3, and 3.9 min for IS1 and 6.4 min for IS2, respectively. The validated dynamic range was 0.2–20 ng/ml for all three metabolites based on a sample volume of 0.25-ml. The linearity of calibration curves for M1, M2, and M3 in both matrices had a correlation coefficient of ≥0.9984. In whole blood, validation data showed intra-batch (n  = 6) CVs of ≤5.9% and REs between −4.9 and 3.6% and inter-batch (N  = 18) CVs of ≤4.9% and REs between −3.5 and 1.5% for all three metabolites. In human plasma, validation data showed intra-batch (n  = 6) CVs of ≤7.3% and REs between −5.1 and 7.6% and inter-batch (N  = 18) CVs of ≤6.6% and REs between −0.3 and 4.7% for all three metabolites. Extraction recoveries were 72% for M1, 87% for M2, 69% for M3, 79% for IS1, and 74% for IS2 from blood; and 94% for M1, 96% for M2, 98% for M3, 92% for IS1, and 93% for IS2 from plasma. All three metabolites in human blood and plasma were stable for three freeze-thaw cycles, or 24-h ambient storage, or 12 months storage at approximately −80 °C. Extracted samples were stable for at least 50 h at room temperature (RT). This method has been successfully used to analyze whole blood and plasma samples from human pharmacokinetic studies. Several key factors affecting the performance of the assay methods have also been addressed briefly.
Keywords: Tacrolimus (FK506) metabolites; Human blood and plasma; Ammonium-adduct LC–MS/MS;

An automated HPLC method with column switching is described for the determination of quetiapine, clozapine, perazine, olanzapine and metabolites in blood serum. After clean-up on silica C8 material (20 μm particle size) drugs were separated on ODS Hypersil C18 material (5 μm; column size 250 mm × 4.6 mm i.d.) within 25 min and quantified by ultraviolet (UV) detection at 254 nm. The limit of quantification ranged between 10 and 50 ng/ml. At therapeutic concentrations of the drugs, the inter-assay reproducibility was below 10%. Analyses of drug concentrations in serum of 75–295 patients treated with therapeutic doses of the antipsychotic drugs revealed mean ± S.D. steady state concentrations of 139 ± 136 ng/ml for quetiapine, 328 ± 195 ng/ml for clozapine, 48 ± 27 ng/ml for olanzapine and 71 ± 52 ng/ml for perazine. The method was thus suitable for routine therapeutic drug monitoring and may be extended to other drugs.
Keywords: Quetiapine; Clozapine; Norclozapine; Olanzapine; Perazine; High-performance liquid chromatography; Ultraviolet detection; Column-switching; TDM; Therapeutic drug monitoring;

A high-performance liquid chromatographic and an UV spectrophotometric method were developed and validated for the quantitative determination of three antiretroviral drugs viz. Lamivudine, Stavudine and Nevirapine that constitute one of the first line regimens in antiretroviral therapy. The different analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2B guidelines. Chromatography was carried out by isocratic technique on a reversed-phase C-18 SYMMETRY column with mobile phase based and optimized depending on the polarity of the molecules. The UV spectrophotometric determinations were performed at 270, 265 and 313 nm for Lamivudine, Stavudine and Nevirapine, respectively. The linearity of the calibration curves for each analyte in the desired concentration range is good (r 2  > 0.999) by both the HPLC and UV methods. Both the methods were accurate and precise with recoveries in the range of 97 and 103% for all the three drugs and relative standard deviation (R.S.D.) <5%. Moreover, the accuracy and precision obtained with HPLC correlated well with the UV method which implied that UV spectroscopy can be a cheap, reliable and less time consuming alternative for chromatographic analysis. The proposed methods are highly sensitive, precise and accurate and hence were successfully applied for the reliable quantification of API content in the commercial formulations of Lamivudine, Stavudine and Nevirapine.
Keywords: Lamivudine; Stavudine; Nevirapine; UV spectrophotometry; RP-HPLC;

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile–phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.
Keywords: On-line derivatization method; Reverse phase chromatography; HPLC; Azithromycin; Serum; Macrolide antibiotics; FMOC-Cl;

Simple and sensitive determination of free and total morphine in human liver and kidney using gas chromatography–mass spectrometry by Keiko Kudo; Tomomi Ishida; Naoki Nishida; Naofumi Yoshioka; Hiromasa Inoue; Akiko Tsuji; Noriaki Ikeda (359-363).
We developed a reliable, simple and sensitive method to determine free and total morphine in human liver and kidney, using gas chromatography–mass spectrometry (GC–MS). Free morphine or total morphine obtained by acid hydrolysis from 0.2 g tissue sample was extracted using an Extrelut® NT column with an internal standard, dihydrocodeine, followed by trimethylsilylation. The derivatized extract was submitted to GC–MS analysis of EI-SIM mode. The calibration curves of morphine in both liver and kidney samples were linear in the concentration range from 0.005 to 5 μg/g. The lower limits of detection of morphine were 0.005 μg/g. This method proved successful when we determined free and total morphine in liver and kidney obtained from an autopsied man who was mis-ingested morphine compound in the hospital, which resulted in the cause of death being morphine intoxication.
Keywords: Morphine; Total morphine; Gas chromatography–mass spectrometry;

Indomethacin (IND) is the drug of choice for the closure of a patent ductus arteriosus (PDA) in neonates. This paper describes a simple, sensitive, accurate and precise microscale HPLC method suitable for the analysis of IND in plasma of premature neonates. Samples were prepared by plasma protein precipitation with acetonitrile containing the methyl ester of IND as the internal standard (IS). Chromatography was performed on a Hypersil C18 column. The mobile phase of methanol, water and orthophosphoric acid (70:29.5:0.5, v/v, respectively), was delivered at 1.5 mL/min and monitored at 270 nm. IND and the IS were eluted at 2.9 and 4.3 min, respectively. Calibrations were linear (r  > 0.999) from 25 to 2500 μg/L. The inter- and intra-day assay imprecision was less than 4.3 % at 400–2000 μg/L, and less than 22.1% at 35 μg/L. Inaccuracy ranged from −6.0% to +1.0% from 35 to 2000 μg/L. The absolute recovery of IND over this range was 93.0–113.3%. The IS was stable for at least 36 h when added to plasma at ambient temperature. This method is suitable for pharmacokinetic studies of IND and has potential for monitoring therapy in infants with PDA when a target therapeutic range for IND has been validated.
Keywords: Indomethacin; Patent ductus arteriosus; Premature neonates; HPLC;

Sensitive and selective determination of valproic acid in plasma by high-performance liquid chromatography (HPLC) is usually achieved with pre-column derivatization. In the present work, the derivatization is omitted due to using a simple but highly selective plasma extraction procedure and an optimized chromatographic condition. Valproic acid and the internal standard octanoic acid were extracted from plasma samples with n-hexane under acidic condition followed by back-extraction into diluted triethylamine. Chromatography was performed on a CN column (250 × 4.6 mm, 5 μm) under isocratic elution with acetonitrile–40 mM aqueous sodium dihydrogen phosphate (30:70, v/v), pH 3.5. Detection was made at 210 nm and analyses were run at a flow-rate of 1 ml/min. The method was specific and sensitive with a quantification limit of 1.25 μg/ml and a detection limit of 0.1 μg/ml in plasma. The mean absolute recovery for valproic acid using the present plasma extraction procedure was 75.8%. The intra- and inter-day coefficient of variation and percent error values of the assay method were all in acceptable range. Calibration curves were linear (r  > 0.999) from 1.25 to 320 μg/ml in plasma.
Keywords: Valproic acid;

A comparison study on fluoxetine (FL) and norfluoxetine (NORFL) quantitation in human plasma was carried out between the recently developed liquid chromatographic method with fluorescence detection (LC–FLD) and an earlier established liquid chromatography–mass spectrometry (LC–MS) laboratory procedure. Comparative method evaluation was based on the analysis of plasma samples obtained from Parkinsonian patients receiving 20 mg of FL per day. The LC–FLD method involves a two-step liquid extraction procedure without any derivatization, followed by direct chromatography on a Zorbax C8 reversed-phase column. The analytical results are discussed in terms of the method validation and the corresponding experimental protocol (r  ≥ 0.998; CV < 9%; LOQ 20 μg/l). There was good correlation between FL, as well as NORFL, plasma levels as determined by the LC–MS and LC–FLD techniques (r  = 0.9597, N  = 16 and r  = 0.9852, N  = 14 for FL and NORFL, respectively). The results confirm that direct FL/NORFL fluorimetric determination is acceptable for routine use in pharmacokinetic and clinical studies.
Keywords: Fluoxetine; Norfluoxetine; Liquid chromatography; Fluorescence detection; Mass spectrometry; Human plasma; Pharmacokinetic analysis;

Author Index (377-380).

Keyword Index (381-390).