Journal of Pharmaceutical and Biomedical Analysis (v.38, #1)

Estimation of log  P ow values for neutral and basic compounds by microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (μMEEKC-IFD) by Jian Tu; H. Brian Halsall; Carl J. Seliskar; Patrick A. Limbach; Francisco Arias; Kenneth R. Wehmeyer; William R. Heineman (1-7).
Microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (μMEEKC-IFD) was used to obtain log  P octanol/water (log  P ow) values for neutral and basic compounds. Six compounds, with log  P ow values between 0.38 and 5.03, were used to create a calibration curve relating the log of retention factors (log  k) obtained from μMEEKC-IFD with the known log  P ow values. The log  P ow values for six additional compounds were determined using the log  k values obtained by μMEEKC-IFD and the linear relationship between log  P ow and log  k established for the standard compounds. The μMEEKC-IFD buffer was composed of 50 mM 3-[cyclohexylamino]-1-propane-sulfonic acid (CAPS) buffer (pH 10.4) containing 1.2% n-heptane (v/v), 2% sodium dodecylsulfate (w/v), 8% 1-butanol (v/v) and 4 μM 5-carboxytetramethyl-rhodamine (TAMRA) as the fluorophore probe for indirect detection. The μMEEKC-IFD provided an accurate method for estimating log  P ow values and also a means for analyzing compounds that are non-fluorescent.
Keywords: Multiplexed capillary electrophoresis; pK a determination; High throughput;

A capillary electrophoresis method for determining the enantiomeric purity of moxifloxacin hydrochloride in drug substance and ophthalmic/otic drug products was developed and validated. Because moxifloxacin hydrochloride has two chiral centers, the existence of four different isomers is possible. The method was capable of separating moxifloxacin hydrochloride, which is the S,S-isomer, from its potential chiral degradation products, which are the R,R-enantiomer, the R,S-diastereomer, and the S,R-diastereomer. The separation was carried out at 20 °C in a 50 μm × 40 cm fused-silica capillary with an applied voltage of −13 kV using a 12.5 mM TEA phosphate buffer (pH 2.5) containing 5% highly-sulfated gamma-cyclodextrin (HS-γ-CD) and 6% acetonitrile. The detection wavelength was 295 nm. The method was validated with respect to its specificity, linearity, range, accuracy, and precision in the expected range of occurrence for the isomeric impurities (0.05–5%). The method was used to investigate whether racemization was a potential degradation pathway for the drug substance, both in the solid state and in solution.
Keywords: Moxifloxacin; Fluoroquinolones; Capillary electrophoresis; Validation; Enantiomeric purity;

A simple, reliable and selective square wave anodic stripping (SWAS) voltammetric method at carbon paste electrode (CPE) of metoclopramide hydrochloride (MCP) in pharmaceutical dosage forms (tablet) and in biological fluids (spiked and real urine samples) has been developed and evaluated. Different parameters such as medium, supporting electrolyte, pH, accumulation potential, scan rate, accumulation time and ionic strength, were tested to optimize the conditions for the determination of MCP. The adsorbed form is oxidized irreversibly under optimal conditions, viz., 0.4 M HCl–sodium acetate buffer (pH ∼ 6.2), 0.2 M KCl, a linear concentration ranges from 0.067 to 0.336, 0.067 to 0.269 and 0.067 to 0.269 ng/mL of MCP, at accumulation times 60, 120 and 180 s, respectively, can be determined successfully. The interferences of some common excipients and some metal ions were studied. The standard addition method was used to determine the MCP in pure solutions, tablets and in biological fluids with satisfactory results. The data obtained are compared with the standard official method.
Keywords: Square wave voltammetry; Carbon paste electrode; Metoclopramide hydrochloride; Tablet; Urine;

Even nowadays millions of people suffer and even die each year from malaria and hundreds of millions of people especially in tropical countries.Quinine (Q) a natural occurring alkaloid and chloroquine (CQ) a synthetic drug are widely used as anti-malarial agents. Herein an isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method is described for the simultaneous determination of quinine and chloroquine, at low concentrations, in pharmaceuticals and biological fluids. The present method is characterized by higher sensitivity and analytes are separated in less time than the already published methods. The analytical column, an MZ Kromasil, C18, 5 μm, 250 × 4 mm, was operated at ambient temperature with backpressure values of 230 kg/cm2. Mobile phase consisted of methanol–acetonitrile–0.1 mol/L ammonium acetate, (45:15:40 v/v) at a flow rate of 1.0 mL/min.Fluorescence detection was performed at excitation 325 nm and emission 375 nm, respectively. Salicylic acid was used as internal standard at a concentration of 0.5 ng/μL, resulting in a detection limit of 0.3 ng, while upper limit of linear range was 0.7 ng/μL for quinine and 0.5 ng/μL for chloroquine. Separation was completed within 5 min.The statistical evaluation of the method was examined performing intra-day (n  = 8) and inter-day calibration (n  = 8) and was found to be satisfactory, with high accuracy and precision results. Solid phase extraction provided high relative extraction recoveries from biological matrices: 92.1% for quinine and 105.4% for chloroquine from blood serum and 101.8% for quinine and 90.7% for chloroquine from urine.
Keywords: Quinine; Chloroquine; Anti-malarial agents; Pharmaceuticals; Biological fluids; Blood serum; Urine; HPLC; SPE;

The study describes a flow injection on-line microdialysis system for in vivo monitoring of ergometrine maleate in rabbit blood with fluorescence detection. A flow-through microdialysis probe was used for intravenous sampling by pumping of the blood from the tested rabbit through the flow-through microdialysis probe located outside the living system at a flow rate of 15 μl min−1. The perfusion rate is 5 μl min−1. The ergometrine maleate in the dialysate was detected on-line with a flow injection fluorescence system after the ergometrine maleate administration (0.2 mg kg−1, i.v.). The dialysate sample volume was about 15 μl. The system was linearly related to the concentration of ergometrine maleate in the range 1–140 ng ml−1 (r  = 0.9989) with a detection limit 0.3 ng ml−1 (3σ). The pharmacokinetic parameters of ergometrine maleate were calculated utilizing the pharmacokinetic software ‘NDST-21’ by a one-compartmental open model.
Keywords: Ergometrine maleate; Microdialysis; In vivo; Fluorescence; Pharmacokinetics;

A sensitive and specific method using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the analysis of total ribavirin in monkey liver is developed and validated. In this method, ribavirin and its phosphorylated metabolites are extracted with perchloric acid. The metabolites are converted to ribavirin using acid phosphatase and further purified using a NH2 solid-phase extraction (SPE) cartridge prior to LC–MS/MS analysis. [13C]Ribavirin is added with the extraction solution as an internal standard to obtain better accuracy and precision of the analysis. The MS/MS was selected to monitor 245 → 113 and 250 → 113 transitions using positive electrospray ionization for ribavirin and [13C]ribavirin. The calibration curve is linear over a concentration of 1.0–100 μg/g with a limit of quantitation (LOQ) of 1.0 μg/g. Mean inter-assay accuracy for QC at 1.0, 10 and 100 μg/g are 108, 99.7 and 99.7%, respectively. Mean inter-assay precision (CV) for QC at 1.0, 10 and 100 μg/g are 5.34, 5.24 and 4.59%, respectively. Extractability of total ribavirin from liver has been confirmed with liver obtained from monkey dosed with [14C]ribavirin. The method has been proven to be useful in the determination of total ribavirin concentration in liver from monkeys in mass balance study (10 mg/kg) and in 28 days toxicology study (300 mg/kg/day). It is also used to determine the total ribavirin concentration in human livers from hepatitis C patients received dose of 600 mg ribavirin twice daily.
Keywords: Ribavirin; Nucleoside; Nucleotide; Hepatitis C; LC–MS/MS; Human liver biopsy;

A validated HPTLC method for determination of tea tree oil from cosmeceutical formulations by S.S. Biju; Alka Ahuja; M.R.M. Rafiullah; Roop. K. Khar (41-44).
A HPTLC method has been developed and validated for the determination of tea tree oil from cosmeceutical formulations. Tea tree oil concentration was estimated by analyzing the terpinen-4-ol content. The method employed TLC aluminium plates precoated with silica gel 60F-254. The solvent system consisted of toluene and ethyl acetate in the ratio 85:15. The calibration curve of terpinen-4-ol was linear in the range of 100–900 ng. The polynomial regression data for the calibration plots showed a good linear relationship with r 2  = 0.9949. The Rf value of terpinen-4-ol was found to be 0.62 ± 0.05. The method was validated for precision and accuracy. The minimum detectable amount was found to be 60 ng. The limit of quantitation was found to be 100 ng. The drug content was within the limits (±5% of the labeled content of the formulations). The recovery of tea tree oil was greater than 99%. The method was found to be simple, sensitive, precise, accurate and specific for estimation of tea tree oil from formulations.
Keywords: HPTLC; Tea tree oil; Terpinen-4-ol; Melaleuca alternifolia;

Simultaneous quantification of six major active saponins of Panax notoginseng by high-performance liquid chromatography-UV method by Lie Li; Jin-lan Zhang; Yu-xin Sheng; De-an Guo; Qiao Wang; Hong-zhu Guo (45-51).
A simple, sensitive and specific high-performance liquid chromatography-UV (HPLC-UV) method has been developed for the first time to simultaneously quantify the six major active saponins of Panax notoginseng, namely notoginsenoside R1, ginsenoside Rg1, Rb1, Rg2, Rh1 and Rd. Astragaloside IV is used as the internal standard. This HPLC assay was performed on a reversed-phase C18 column with gradient elution of acetonitrile and 0.01% formic acid in 30 min. The method provided good reproducibility and sensitivity for the quantification of six saponins with overall intra- and inter-day precision and accuracy of less than 4.0% and higher than 90%, respectively. This assay is successfully applied to the determination of the six saponins in 23 notoginseng samples. The results indicated that the developed HPLC assay can be readily utilized as a quality control method for P. notoginseng.
Keywords: High-performance liquid chromatography; Panax notoginseng; Ginsenosides; Notoginsenoside;

Assay validation for three antidepressants in pharmaceutical formulations: Practical approach using capillary gas chromatography by J.J. Berzas Nevado; M.J. Villaseñor Llerena; A.M. Contento Salcedo; E. Aguas Nuevo (52-59).
An easy and fast capillary gas chromatographic FID method, which was already described by the same authors for the simultaneous determination of fluoxetine, fluvoxamine and clomipramine without derivatization step, is now submitted to a validation procedure in several pharmaceutical formulations. Main aspects of the validation method are examined and discussed, since methods for regulatory submission in most cases must demonstrate: specificity in presence of all potential components, concentration range over which the response is lineal, accuracy, precision, acceptable detection and quantitation limits and stability of the procedure. The pharmaceutical preparations subject of validation were: ‘Prozac’ (capsules), ‘Dumirox’ (tablets) and ‘Anafranil’ (tablets) containing fluoxetine, fluvoxamine and clomipramine, respectively. The results presented in this report show the applied gas chromatographic method is acceptable for the determination of the three antidepressants in the pharmaceutical formulations above mentioned.
Keywords: Gas chromatography; Validation; Drug analysis; Fluoxetine; Fluvoxamine; Clomipramine; Antidepressants; Selective serotonin reuptake inhibitors;

A simple and novel method for the extraction and quantification of diltiazem hydrochloride was developed and applied to homogenization and stability studies. The method used solid phase extraction coupled with reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Validation showed inter-day recoveries ranging from 84.00 to 96.52% with relative standard deviations ranging from 12.01 to 15.94%. Intra-day recoveries ranged from 67.95 to 106.1% with relative standard deviations less than 5%. The method showed excellent linearity from 50 to 250 mg/ml in undiluted gel (R 2  = 0.996). The homogenization study showed good homogenization using both 50 and 100 depression techniques. Diltiazem was stable at a concentration of 246 mg/ml for 30 days and at a concentration of 99.6 mg/ml for 60 days no matter the storage conditions explored in this study.
Keywords: Diltiazem; Reverse-phase liquid chromatography; Transdermal gel; Drug stability; Compounded drug analysis;

A capillary zone electrophoresis method for the separation and determination of thiamin and its phosphate esters (free thiamin, thiamin monophosphate, and thiamin pyrophosphate) was developed and optimized. The efficiency achieved with boric acid–sodium tetraborate decahydrate buffer (pH 8.24; 65–8 mM) at an applied potential of 30 kV gave the detection limit (S/N = 3) and the limit of quantitative measurement (S/N = 10) of thiamin and its phosphate esters ranging from 10−4 to 6 × 10−4  mM and from 6 × 10−4 to 1.2 × 10−3  mM, respectively. The effects of pH on separation and migration times of thiamin and its phosphate esters are described. The method was validated and applied to the quantitative determination of thiamin in commercial tablets containing both a massive and a normal dose of thiamin.
Keywords: Thiamin esters; Separation; Capillary zone electrophoresis;

Stability-indicating methods for determination of vincamine in presence of its degradation product by Mostafa A.M. Shehata; Mohammad A. El Sayed; Mohammad F. El Tarras; Mohammad G. El Bardicy (72-78).
Three different stability indicating assay methods are developed and validated for determination of vincamine in the presence of its degradation product (vincaminic acid). The first method is based on the derivative ratio zero crossing spectrophotometric technique using 0.1 N hydrochloric acid as a solvent. In the second method, measurements are based on spectro-densitometric technique using high performance thin-layer chromatography (HPTLC) plates with a developing system consisting of methanol–chloroform–ethyl acetate (2:1:1, v/v/v). The third method depends on high-performance liquid chromatography (HPLC). Separation of vincamine from vincaminic acid using Lichrocart RP-18 column (250 mm × 4.6 mm i.d.) with a mobile phase consisting of acetonitrile–ammonium carbonate (0.01 M) (7:3, v/v) is achieved. The methods showed high sensitivity with good linearity over the concentration ranges of 12 to 48 μg ml−1, 3 to 17 μg/spot, and 2 to 20 μg ml−1 for derivative spectrophotometry, spectro-densitometry and HPLC methods, respectively. The developed methods were successfully applied to the analysis of pharmaceutical formulations containing vincamine with excellent recoveries.
Keywords: Vincamine; Vincaminic acid; Derivative ratio spectrophotometry; Densitometry; Liquid chromatography;

LC–ESI-MS/MS characterization of strophanthin-K by Giorgio Grosa; Gianna Allegrone; Erika Del Grosso (79-86).
A liquid chromatography–mass spectrometry (LC–MS) method was developed for the characterization of strophanthin-K, a mixture of cardiac glycosides extracted from the seeds of Strophanthus kombè. The method is based on the separation of the cardenolides using high performance liquid chromatography (HPLC) followed by detection with electrospray ionization mass-spectrometry (ESI-MS). Chromatographic separation of the analytes was achieved on a RP C-18 column using water: 1% formic acid in water (v/v):acetonitrile gradient mobile phase. Strophanthin-K glycosides studied in ESI-MS in negative ion mode formed abundant adduct ions [M  + HCOO] while the pseudomolecular ions [M  − H] are obtained in ESI-MS/MS experiments. Six different cardiac glycosides were identified and characterized: k-strophanthoside, k-strophanthin-β, helveticoside (erysimin), erysimoside, cymarin and neoglucoerysimoside.Forced degradation investigations done with strophanthin-K showed that k-strophanthidin (the aglycone of strophanthin-K glycosides) was the main product of degradation in acidic conditions; however, in basic conditions, the hydrolysis of the unsaturated 17β-lactones to the corresponding γ-hydroxy acids was the predominant degradation pathway.
Keywords: Strophanthin-K; k-Strophanthoside; Cardiac glycosides; Cardenolides; LC–ESI-MS/MS;

The migration behaviour of acetaminophen and p-aminophenol was investigated by capillary electrophoresis. The influence of different parameters (pH, nature and concentration of the running buffer and applied voltage) on the migration time, peak symmetry, efficiency and resolution was systematically investigated. The two analytes can be well separated within 4 min in a 57 cm fused-silica capillary at a separation voltage of 18 kV in a 50 mM borate buffer adjusted to pH 9.5. Correlation coefficients for calibration curves in the range 0.2–200 μg ml−1 for acetaminophen and 0.3–3 μg ml−1 for p-aminophenol were higher than 0.999. The sensitivity of detection is 4.2 ng ml−1 for acetaminophen and 11.2 ng ml−1 for p-aminophenol. The method was applied to the analysis of various commercially available acetaminophen dosage forms with recoveries of 98.4–100.7%.
Keywords: Acetaminophen; p-Aminophenol; Capillary zone electrophoresis; Pharmaceuticals;

In this work, the artificial neural networks (ANN) and partial least squares (PLS) were applied to data obtained by differential pulse voltammetry for the determination of vitamins in synthetic and pharmaceutical samples. For calibration purposes, both synthetic and commercial samples were employed as standards. From the results it was possible to verify that ANN is the best method for modeling the data due to the fact that interactions among electro-active components result in non-linear response on the glassy carbon electrode. The results achieved for the determination of vitamins in pharmaceutical samples using ANN method provided a maximum value for relative error of 0.40% for VC, 8.3% for VPP and 9.1% for VB6. The proposed methodology is simple, rapid and can be easily used to control quality laboratories as an alternative analysis method.
Keywords: Vitamin C; Vitamin B6; Vitamin PP; Differential pulse voltammetry; Partial least squares; Artificial neural networks;

A flow-injection chemiluminescence method for the determination of tryptophan was proposed, which was based on an intense chemiluminescence of tryptophan in hydrogen peroxide–nitrite–sulfuric acid medium. The chemiluminescence reaction was attributed to peroxidation and epoxidation of tryptophan by peroxynitrous acid, and subsequent decomposition of the formed dioxetane. The chemiluminescence intensity was linear with tryptophan in the range of 6.0 × 10−7 to 3.0 × 10−5  mol l−1 and the limit of detection (S/N = 3) was 1.8 × 10−7  mol l−1. The proposed method was applied to the analysis of tryptophan in pharmaceutical preparations and human serum.
Keywords: Chemiluminescence; Peroxynitrous acid; Flow-injection; Tryptophan;

Liquid chromatography/tandem mass spectrometry for the determination of magnesium lithospermate B in beagle dog serum by Xiaochuan Li; Chen Yu; Weikang Sun; Jie Lu; Lijiang Xuan; Yiping Wang (107-111).
A sensitive and specific isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of magnesium lithospermate B (MLB) in beagle dog serum with silibinin as internal standard. The serum samples were treated by special liquid–liquid extraction, and the analytes were determined using electrospray negative ionization mass spectrometry in the selective monitoring mode, with sufficient sensitivity to allow analysis of dog serum samples generated following administration of a clinically relevant dose. The calibration curve for MLB was linear over the range 8–2048 ng/ml with coefficients of correlation >0.999. The intra- and inter-day precisions (CV) of analysis were <7%, and accuracy ranged from 90 to 106%. This quantitation method was successfully applied to a pharmacokinetic study of i.h. administration of MLB with dosages of 3, 6, 12 mg/kg in beagle dogs.
Keywords: LC/MS/MS; Pharmacokinetics; MLB; Beagle dogs;

LC–MS method for the estimation of Δ8-THC and 11-nor-Δ8-THC-9-COOH in plasma by Satyanarayana Valiveti; Dana C. Hammell; D. Caroline Earles; Audra L. Stinchcomb (112-118).
The aim of the present study was to develop a simple and sensitive LC–MS method for the estimation of Δ8-tetrahydrocannabinol (Δ8-THC) and its metabolite, 11-nor-Δ8-tetrahydrocannabinol-9-carboxylic acid (11-nor-Δ8-THC-9-COOH), in guinea pig plasma after topical drug application. The plasma samples were analyzed by LC–MS using negative-mode electrospray ionization detection and a simple liquid–liquid extraction technique. The mean recoveries for Δ8-THC and its metabolite, 11-nor-Δ8-THC-9-COOH, were 96.6 and 88.2%, respectively. The lower limits of quantification (LLOQ) for Δ8-THC and 11-nor-Δ8-THC-9-COOH were 3.97 and 7.26 nM, respectively. The topical treatment steady-state plasma concentrations of Δ8-THC and 11-nor-Δ8-THC-9-COOH were 8.24–27.63 and 19.66–23.17 nM, respectively, with a lag period of 0.3–2.2 h. This assay method is selective, sensitive, and reproducible for the determination of Δ8-THC and 11-nor-Δ8-THC-9-COOH at low concentrations in small volumes of plasma.
Keywords: Δ8-THC; Δ8-THC-9-COOH; Guinea pig plasma; Topical application; LC–MS;

Liquid chromatography/tandem mass spectrometry for the determination of carbamazepine and its main metabolite in rat plasma utilizing an automated blood sampling system by Yongxin Zhu; Hwa Chiang; M. Wulster-Radcliffe; Rita Hilt; Philip Wong; Candice B. Kissinger; Peter T. Kissinger (119-125).
A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of carbamazepine and its main metabolite carbamazepine 10,11-epoxide in rat plasma is described. The method consists of a liquid–liquid extraction procedure and electrospray LC/MS/MS analysis. The chromatographic separation was achieved within 5 min using a C8 (150 mm × 2.1 mm) 5 μm column with a mobile phase composed of water/acetonitrile/acetic acid (69.5:30:0.5, v/v/v) at a flow rate of 0.4 ml/min. D10-carbamazepine is used as the internal standard for all compounds. Analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode using selected reaction monitoring (SRM). Carbamazepine was monitored by scanning m/z 237 → 194, carbamazepine 10,11-epoxide by m/z 253 → 210 and d10-carbamazepine by m/z 247 → 204. The lower limit of quantitation (LLOQ) is 5 ng/ml for each analyte, based on 0.1 ml aliquots of rat plasma. The extraction recovery of analytes from rat plasma was over 87%. Intra-day and inter-day assay coefficients of variations were in the range of 2.6–9.5 and 4.0–9.6%, respectively. Linearity is observed over the range of 5–2000 ng/ml. This method was used for pharmacokinetic studies of carbamazepine and carbamazepine 10,11-epoxide in response to two different blood sampling techniques (i.e., manual sampling versus automated sampling) in the rat. Several differences between the two sampling techniques suggest that the method of blood collection needs to be considered in the evaluation of pharmacokinetic data.
Keywords: Carbamazepine; Metabolite; LC/MS/MS; Autosampling;

Ginseng saponins isolated from ginseng, have been regarded as the principal constituents responsible for the biological activities. The aim of this study was to set up a liquid chromatography–mass spectrometry (LC–MS) method for simultaneously determine the concentration of Ginsenoside Rg1 and its secondary glycoside Rh1 and aglycone protopanaxatriol (PPT) in rat plasma so as to study the pharmacokinetics of Rg1 after intraveneous (i.v.) and intragastric gavage (i.g.) administration. One hundred microliters or 1.0 ml of rat plasma samples from i.v. or i.g. treated rats were used respectively for analysis. After solid-phase extraction (SPE) and high performance liquid chromatography (HPLC) separation, the chloride adduct anions [M + Cl] of Rg1, Rh1 and PPT were analyzed by LC–MS in selected ions monitoring (SIM) mode. Rg1 could be determined by this LC–MS method over the ranges of 1.56–250 ng/ml and 250–20,000 ng/ml with the correlation coefficients of 0.999 and 0.9998, respectively. The detection limits (LOD) of this method was 20 pg (S/N>3) for Rg1, 100 pg for Rh1 and 10 pg for PPT. Chromatographic separation was achieved in less than 8 mins. The method has been used for the pharmacokinetic study of Rg1 in rats.
Keywords: Saponin; Ginsenosides; Rg1; Rh1; PPT; LC–MS; SPE;

Application of capillary zone electrophoresis in the separation and determination of the curcuminoids in urine by Kailong Yuan; Qianfeng Weng; Hongying Zhang; Jianhui Xiong; Guowang Xu (133-138).
The major components of the plant curcuma longa are the curcuminoids that include curcumin, demethoxycurcumin and bisdemethoxycurcumin. It has been reported the curcuminoids have some important activities. A new CZE method with diode array detection has been developed for the separation and determination of the curcumin, demethoxycurcumin and bisdemethoxycurcumin. Three curcuminoids could be readily separated within 7 min with a 15 mM sodium tetraborate buffer containing 10% methanol (v/v) at pH 10.8, 25 kV and 30 °C. The method has been validated and shows good performance with respect to selectivity, reproducibility, linearity, limits of detection and recovery. The proposed method was successfully applied to determine the curcuminoids in urine.
Keywords: Curcuminoids; Capillary zone electrophoresis; Urine; Curcuma longa;

Bioanalysis of HIV protease inhibitors in samples from sanctuary sites by K.M.L. Crommentuyn; A.D.R. Huitema; J.H. Beijnen (139-147).
The human immunodeficiency virus (HIV) is present in several sites inside the human body, which are hardly accessible to antiretroviral drugs, the so-called sanctuary sites. The most important sanctuary sites are cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMCs) and seminal plasma. The determination of drug concentrations in these sanctuary sites may form an important step in treatment optimisation of HIV-infected individuals. However, bioanalysis in these sites is hampered by several factors with regard to sample preparation, chromatography and detection. In this review, we will discuss these issues and give an overview of published methods using high-performance liquid chromatography (HPLC) for the bioanalysis of HIV protease inhibitors in CSF, PBMCs and seminal plasma.
Keywords: Bioanalysis; HPLC; HIV protease inhibitors; PBMC; CSF; Seminal plasma;

A method of analysis of pinosylvin in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in natural products. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of pinosylvin and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL) were precipitated with acetonitrile after addition of the internal standard, 7-ethoxycoumarin. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 mm × 4.6 mm, ID, 5 μm) with UV detection at 308 nm. The calibration curves were linear ranging from 0.5 to 100 μg/mL. The mean extraction efficiency was >99%. Precision of the assay (coefficient of variation) was <10%, including the limit of quantitation (0.5 μg/mL). Bias of the assay was lower than 15%. The limit of detection was 100 ng/mL for a 0.1 mL sample. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of pinosylvin. Three metabolites of pinosylvin, two oxidative and one glucuronidated, have been identified. The two oxidative metabolites of pinosylvin have been identified as E- and Z-resveratrol.
Keywords: Reversed-phase HPLC; UV-detection; Kinetics; Pinosylvin;

Determination of troglitazone stereoisomers in rat plasma using semi-micro HPLC with electrochemical detection by Nobuyuki Suzuki; Naoto Miyashita; Akira Kotani; Fumiyo Kusu; Takao Kawasaki (155-161).
A highly sensitive determination method for troglitazone stereoisomers was developed by high-performance liquid chromatography with electrochemical detection (HPLC–ECD). The oxidation behavior of troglitazone was investigated for the application of ECD by measuring the cyclic voltammogram. The separation was performed on a semi-micro chiral column (Chiralcel OJ-RH) using a mobile phase consisting of methanol–acetic acid (1000:1, v/v) containing 50 mM LiClO4 at a flow rate of 20 μl/min. The peak areas of the stereoisomers separated from 0.1 to 50 ng/ml of troglitazone had good linearity with correlation coefficients of >0.999, and had similar response. The limit of detection was 1.3 fmol (signal-to-noise ratio of 3). This method was applied to the determination of troglitazone stereoisomers in rat plasma. The levels of troglitazone stereoisomers in rat plasma could be monitored until 24 h after the oral administration.
Keywords: Chiral separation; Electrochemical detection; Rat plasma; Semi-micro-HPLC; Troglitazone;

Voltammetric assay of Guaifenesin in pharmaceutical formulation by I. Tapsoba; J.-E. Belgaied; K. Boujlel (162-165).
The electrochemical oxidation of Guaifenesin in a pharmaceutical formulation containing Guaifenesin has been carried out in Britton–Robinson buffer (BRB) (0.04 mol L−1) on platinum electrode. Guaifenesin exhibits a well-defined irreversible oxidation peak at 0.924 V/ref. The influence of pH on the oxidation of Guaifenesin was studied in BRB (pH range 2-5). A method for the analysis of Guaifenesin in BRB (0.04 mol L−1, pH 2), which allows quantification over the range 20–60 μg mL−1, was proposed and successfully applied to the determination of Guaifenesin in syrup with mean recovery and relative standard deviation of 103.3% and 1.32%, respectively.
Keywords: Gauifenesin; Oxidation; Cyclic voltammetry; Platinum electrode; Irreversible;

Determination of β2-agonists by ion chromatography with direct conductivity detection by Suhui Shen; Jin Ouyang; Willy R.G. Baeyens; Joris R. Delanghe; Yiping Yang (166-172).
A simple method for the simultaneous detection of four β2-agonists (salbutamol, fenoterol, clorprenaline, and clenbuterol) using ion chromatography (IC) with direct conductivity detection (CD) based on their ionization in acidic medium without chemical suppression is presented. The mixture of 1.8 mM HNO3 and 2% (v/v) acetonitrile was used as eluent. The method could be applied to the determination of the β2-agonists in pharmaceutical preparations. The recovery of salbutamol and clenbuterol in tablets was more than 97% (n  = 3) and the relative standard deviation (n  = 11) less than 2.8%. With the proposed method, salbutamol could also be successfully detected in human plasma. In a single chromatographic run, the four β2-agonists can be separated and determined in less than 8 min. The linear ranges were of 7.0–1.4 × 103  ng/ml for salbutamol, 34–7.8 × 103  ng/ml for fenoterol, 8.0–1.6 × 103  ng/ml for clorprenaline, and 25–7.5 × 103  ng/ml for clenbuterol. The detection limits were 2.0 ng/ml for salbutamol, 10 ng/ml for fenoterol, 3.0 ng/ml for clorprenaline, and 10 ng/ml for clenbuterol.
Keywords: β2-Agonists; Salbutamol; Fenoterol; Clorprenaline; Clenbuterol; Ion chromatography; Conductivity detection;

A simple, precise and accurate isocratic reverse-phase liquid chromatography method with programmed wavelength detection has been validated to quantify DRF-4367 and Phenol red, simultaneously for application in rat in situ single pass intestinal perfusion study to assess intestinal permeability of DRF-4367, a novel cox-2 inhibitor. The method was validated on RP C-18 analytical column. Mobile phase consisted of sodium dihydrogen orthophosphate (pH 3.2, 0.01 M)–acetonitrile–methanol (30:50:20, v/v/v). The developed method has a short run time of 12 min with a flow rate of 1.0 ml/min. The injector volume was set to 20 μl and acquisition was carried out using a PDA detector while processing was done by timed wavelength extraction. The percentage R.S.D. and recovery in all studies indicated that the method was suitable for the intended purpose. The validated method was found to be linear and precise in the working range. Suitability of storage at cold temperature was ensured along with complete sample recovery.
Keywords: Reverse-phase liquid chromatography; Intestinal permeability; Validation;

Quality control and stability study using HPTLC: applications to cyclophosphamide in various pharmaceutical products by Jérôme Bouligand; Thomas Storme; Isabelle Laville; Lionel Mercier; Odile Oberlin; Gilles Vassal; Philippe Bourget; Angelo Paci (180-185).
Cyclophosphamide is an alkylating agent widely used from cancer chemotherapy to immunotherapy purposes. In paediatrics oncology, oral cyclophosphamide prescribed at low dosages for a long time treatment is currently investigated. This treatment is a putative well tolerated regimen for children treated for a wide variety of recurrent solid tumours. For these purposes, new oral formulations more convenient for children than cyclophosphamide 50 mg tablets are needed. Thus, we present a rapid method for the assay of cyclophosphamide in various pharmaceutical preparations using high-performance thin-layer chromatography (HPTLC) and derivatization with phosphomolybdic acid. This method is accurate and precise and allows quantitation of cyclophosphamide in aqueous solutions from 400 to 1200 μg/mL. It is suitable for quantitation and stability studies of cyclophosphamide in pharmaceutical products, i.e. capsules and infusion bags prepared in a hospital pharmacy. According to pharmaceutical guidelines, we demonstrated that low dose cyclophosphamide capsules, extemporaneously prepared for children, are stable at least for 70 days.
Keywords: Cyclophosphamide; HPTLC; Stability study; Quality control;

Measurement of lactose crystallinity using Raman spectroscopy by Bridget M. Murphy; Stuart W. Prescott; Ian Larson (186-190).
Raman spectroscopy (RS) was used to determine the crystallinity of lactose (a commonly used carrier in dry powder inhaler (DPI) formulations). Samples of α-lactose monohydrate and amorphous lactose were prepared using ethanol precipitation and lyophilisation respectively. The anomeric forms were confirmed using DSC at a rate of 10 °C/min and heated to 250 °C. The Raman spectra of both α-lactose monohydrate and amorphous lactose were obtained. Distinguishable differences were seen between the two spectra including peak areas and intensities. Depolarisation ratios (ρ) of each form were then determined to identify the crystallinity of the lactose carrier samples. At the prominent Raman bands 865 and 1082 cm−1, significant differences in ρ values were observed for crystalline (0.80 ± 0.07, 0.89 ± 0.06 respectively) and amorphous samples (0.44 ± 0.07, 0.51 ± 0.10).
Keywords: Raman spectroscopy; Depolarisation ratio; Lactose; Crystallinity; Anomeric form; Dry powder inhalation;

Electrochemical detection of enzyme labeled DNA based on disposable pencil graphite electrode by Pinar Kara; Arzum Erdem; Stella Girousi; Mehmet Ozsoz (191-195).
Electrochemical biosensor for the detection of DNA hybridization using the reduction signal of α-naphthol is described. A pencil graphite electrode was used as a working electrode. Capture probes were covalently attached on to the pencil graphite electrode surface (PGE) at the 5′ end amino group by using N-(dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent on to PGE. After capture probe immobilization on to PGE surface; probe was hybridized with complementary biotinylated oligonucleotide. Alkaline phosphatase labeled with extravidin (Ex-AP) binds to biotinylated hybrid via biotin–avidin interaction. α-Naphthyl phosphate (α-NAP) was added and the reaction between alkaline phosphatase (AP) and α-NAP was occurred consequently as a substrate of AP, α-NAP reduction signal was obtained from this reaction, at −0.100 V by using differential pulse voltammetry (DPV). Other experimental parameters were studied such as; optimizations of hybridization time, and the concentrations of capture probe, biotinylated oligonucleotide and enzyme.
Keywords: DNA biosensors; α-Naphthol; Alkaline phosphatase; Electrochemistry; Pencil graphite electrode;