Journal of Chromatography B (v.793, #2)

News Section (N1-N3).

Published analytical methods for the quantitative determinations of presently available five 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (“statins”), lovastatin, simvastatin, pravastatin, fluvastatin and atorvastatin, are reviewed for therapeutic drug monitoring purpose in patients. Almost all assay reviewed are based on high-performance liquid chromatography or gas chromatography. Some purification steps (liquid–liquid extraction, solid-phase extraction, etc.) have been used before they are submitted to separation by chromatographic procedures and they are detected by various detection methods like UV, fluorescence and mass spectrometry. This review shows that most method may be used quantitative determination of statins in plasma and they are suitable for therapeutic drug monitoring purpose of these drugs.
Keywords: 3-Hydroxy-3-methylglutaryl;

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine by Jochen Buechler; Matthias Schwab; Gerd Mikus; Beate Fischer; Leo Hermle; Claudia Marx; Georg Grön; Manfred Spitzer; Karl-Artur Kovar (207-222).
An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using β-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (λ ex 286 nm, λ em 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.
Keywords: Ecstasy; N-Ethyl-3,4-methylenedioxyamphetamine;

Determination of urinary hippuric acid by micellar electrokinetic capillary chromatography by Cecilia Zuppi; Diana Valeria Rossetti; Alberto Vitali; Federica Vincenzoni; Bruno Giardina; Massimo Castagnola; Irene Messana (223-228).
We propose a method for the simultaneous determination of hippuric acid (HA) and creatinine based on capillary micellar electrokinetic chromatography. Experimental conditions were 20 mM sodium phosphate, pH 7.20, 25 mM sodium dodecyl sulfate, 5% (v/v) acetonitrile. Electropherograms evidenced HA and creatinine peaks in less than 12 min. The method showed good linearity for both analytes and satisfactory within-day precision. The present method, which is accurate, sensitive, rapid and simple, may be applied to single-spot urine samples.
Keywords: Hippuric acid;

Imaging surface plasmon resonance system for screening affinity ligands by Paul R. Morrill; R.B. Millington; Christopher R. Lowe (229-251).
A surface plasmon resonance (SPR) system for screening ligands for application in affinity chromatography is described. A combinatorial library of 13 ligands was synthesised, characterised and immobilised to agarose beads and gold SPR devices. Binding and elution behaviour and a range of K AX values (103 to 105 M −1) were measured against two target proteins, an insulin analogue (MI3) and a recombinant clotting factor (rFVIIa), in order to create a relational database between the traditional chromatographic format and the new SPR screening system. The SPR transducer surface was fabricated with affinity ligands in a two-dimensional, spatially addressable format, which was durable (>100 cycles) and stable over 6 months. The imaging SPR system comprised a direct optical, CCD-based, instrument capable of imaging the change in refractive index created by biochemical interactions and allowed affinity ligands to be evaluated 15-fold faster with 130-fold less target protein than conventional chromatographic methods. The binding and elution data from both the SPR and chromatographic systems for both target proteins were comparable, with the K AX value generating a nearly linear correlation (R 2=0.875) and a slope bias of ∼2.5±0.25-fold higher for the SPR system. The imaging SPR system has proven capable of screening and evaluating affinity ligands for potential use in the recovery of biopharmaceutical proteins.
Keywords: Affinity ligands;

A sensitive and selective liquid chromatography–tandem mass spectrometry method (LC–MS–MS) for the simultaneous estimation of bulaquine and primaquine has been developed and validated in monkey plasma. The mobile phase consisted of acetonitrile/ammonium acetate buffer (20 mM, pH 6) (50:50 v/v) at a flow-rate of 1 ml/min. The chromatographic separations were achieved on two spheri cyano columns (5 μm, 30×4.6 mm I.D.) connected in series. The quantitation was carried out using a Micromass LC–MS–MS with an electrospray source in the multiple reaction monitoring (MRM) mode. The analytes were quantified from the summed total ion value of their two most intense molecular transitions. This is another novel method leading to increased sensitivity and precision. A simple liquid–liquid extraction with 2×1.0 ml n-hexane/ethyl acetate/dimethyloctyl amine (90:10:0.05, v/v) was utilized. The method was validated in terms of recovery, linearity, accuracy and precision (within- and between-assay variation). The recoveries from spiked control samples were ⩾90 and 50% for bulaquine and primaquine, respectively. Linearity in plasma was observed over a dynamic range of 1.56–400 and 3.91–1000 ng/ml for bulaquine and primaquine, respectively.
Keywords: Bulaquine; Primaquine;

Microemulsion electrokinetic chromatography (MEEKC) using dynamically coated capillary columns is shown to be suitable for estimating the octanol–water partition coefficient (log P) for neutral and weakly acidic compounds at pH 3. The solvation parameter model is used to demonstrate that the retention properties of sodium dodecyl sulfate (1.4% w/v), n-butanol (8% v/v) and n-heptane (1.2% v/v) microemulsion are strongly correlated with the octanol–water partition system. For compounds of varied structure and log P values from 0.3 to 5.15, the correlation model is able to estimate log P to better than 0.25 log units. The dynamically coated columns consisting of a bilayer of poly(vinylsulfonate) adsorbed on top of polybrene provide a suitable electroosmotic flow at pH 3 without interfering in the retention properties of the microemulsion. For automated measurements the microemulsion run buffer should be replenished after 10 runs to maintain a stable cycle time and the coated columns replaced after 40–70 runs, depending on sample properties.

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography, partition and precipitation by Yoichi Shibusawa; Yohko Ino; Takashi Kinebuchi; Mitsuhiro Shimizu; Heisaburo Shindo; Yoichiro Ito (275-279).
Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51–56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS–polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.
Keywords: Proteins, single-strand DNA binding; Single-strand DNA;

Mitoguazone is an antiproliferative agent used in chemotherapy. This study describes a simple and sensitive high-performance liquid chromatographic method for the determination of mitoguazone in 100 μl of plasma. Samples were deproteinized with 100 μl of a solution of internal standard (amiloride, 10 μg/ml) in acetonitrile. An aliquot of the supernatant was injected onto the column. HPLC separation was achieved on a silica column with the mobile phase of methanol–50 mM potassium phosphate buffer (pH 3)–triethylamine (80:20:0.3, v/v), at a flow-rate of 1 ml/min. The eluent was detected at 320 nm. The retention time was about 5.5 min for amiloride and 12 min for mitoguazone. No endogenous substances were found to interfere. Calibration curves were linear from 0.25 to 50 μg/ml. The absolute recoveries of mitoguazone and amiloride were both greater than 84%. The limit of quantitation was 0.25 μg/ml. The intra- and inter-day precision (expressed as RSD) was 5.8%, or less, and the accuracy was 94.7% of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring mitoguazone concentration.
Keywords: Mitoguazone;

A sensitive assay for the determination of (−)-verbenone 10-hydroxylation catalyzed by rat liver microsomes was developed using high-performance liquid chromatography. Verbenone was incubated in vitro with liver microsomes of untreated rats and rats treated with phenobarbital and the products thus formed were extracted with CH2Cl2 and the extracts were separated by HPLC with a C18 5-μm analytical column. Elution was conducted with 40% methanol containing 20 mM NaClO4 and the detection of UV absorbance was done at 251 nm. Product formation was dependent on the incubation time at least up to 30 min and the microsomal protein concentration between 0.01 and 0.1 mg protein/ml. The limit of detection of (−)-10-hydroxyverbenone with the HPLC was found to be about 40 pg, indicating that this method is about 100-fold sensitive than the GC–MS method. Optimized pH for the reaction was at 7.4 when examined with 100 mM potassium phosphate buffer in different pHs. Kinetic analysis showed that K m values for liver microsomes of untreated and phenobarbital-treated rats were 206 and 41 μM and V max values were 5.8 and 44 nmol/min/mg protein, respectively. Thus the present results provided a sensitive and useful method for the determination of verbenone 10-hydroxylation catalyzed by rat liver microsomes.
Keywords: Verbenone; (−)-10-Hydroxyverbenone;

A HPLC–MS–MS method was developed for the determination of the plant lignan 7-hydroxymatairesinol and its potential metabolites matairesinol, oxomatairesinol, α-conidendrin, 7-hydroxyenterolactone, enterodiol, and enterolactone in human plasma. The method included sample cleanup by solid-phase extraction (SPE) and analysis using a PE Sciex API3000 triple quadrupole mass spectrometer with electrospray ionisation. The lignans were quantified using two deuterated internal standards. They showed good chromatographic linearity, analysis repeatability, and SPE recovery in the presence of plasma. In pooled plasma and in plasma samples collected from two individual subjects lignan glucuronides and sulfates were enzymatically hydrolysed to free lignans and then analysed. All the lignans could be detected in the samples.
Keywords: 7-Hydroxyenterolactone; Mammalian lignans; Enterolactone; Enterodiol;

Determination using liquid-chromatography–electrospray tandem mass spectroscopy of ethinylestradiol serum pharmacokinetics in adult Sprague–Dawley rats by Nathan C. Twaddle; Mona I. Churchwell; Retha R. Newbold; K.Barry Delclos; Daniel R. Doerge (309-315).
The pharmacokinetics of ethinylestradiol (EE2), a potent synthetic estrogen, was investigated in male and female Sprague–Dawley rats as part of a series of endocrine-active compounds, including genistein and nonylphenol. A method based on solid-phase extraction and LC with negative ion electrospray tandem mass spectrometric detection was developed and validated. The limit of detection in untreated rat serum was below 0.01 ng/ml (0.03 nM), the limit of quantification was 0.03 ng/ml (0.10 nM), the intra- and inter-day precision was 2–9%, and the intra- and inter-day accuracy was 89–94%. This method was used to determine the serum pharmacokinetics of EE2 in rats following oral gavage administration of 1 mg/kg body weight. EE2 was present in serum primarily in the unconjugated form at concentrations below 0.5 ng/ml. The maximal serum concentration was proportional to dose over the range of 0.04–0.5 mg/kg body weight and pharmacokinetic parameters were determined using model-independent analysis. Significant sex differences were observed for elimination half-times and volumes of distribution, but not for total serum clearance or maximal concentrations. The pharmacokinetic analysis of EE2 will be useful for comparing the toxicological effects of EE2 to those of other environmental estrogens in related rodent endocrine disruptor studies.
Keywords: Ethinylestradiol;

Sensitive determination of tenofovir in human plasma samples using reversed-phase liquid chromatography by S. Sentenac; C. Fernandez; A. Thuillier; P. Lechat; G. Aymard (317-324).
A new high-performance liquid chromatography assay was developed for the determination of tenofovir, a nucleotide analogue, in plasma. A solid–liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase containing Na2HPO4 buffer, tetrabutylammonium hydrogen sulfate and acetonitrile for different elution through a C18 column with UV detection. The method proved to be accurate, precise and linear between 10 and 4000 ng/ml. The method was applied to determine trough levels of tenofovir in 11 HIV-infected patients with virologic failure under multiple antiretroviral therapy. This method was also successfully applied to a pharmacokinetic study in an HIV infected patient with renal failure.
Keywords: Tenofovir;

Novel purification system for 6xHis-tagged proteins by magnetic affinity separation by André Frenzel; Christian Bergemann; Gabi Köhl; Thomas Reinard (325-329).
We have developed a novel nickel–silica matrix for the generation of magnetic beads for metal-ion affinity chromatography. In contrast to magnetic Ni–NTA agarose beads, the novel particle type (SiMAC) consists of a magnetic core and a nickel–silica composite matrix with the nickel ions tightly integrated in the silica. This results in a much higher number of chelating groups compared with Ni–NTA agarose beads. With the SiMAC beads, greatly improved purification of histidine-tagged proteins from crude bacterial extracts was achieved. The yield was at least twice as high as with conventional materials, the method is faster, since the coupling step is omitted and there is no need for handling toxic Ni2+ salts.
Keywords: Proteins, 6xHis-tagged;

R,S-4′-Methoxy-α-pyrrolidinopropiophenone (MOPPP) is a new designer drug with assumed amphetamine-like effects, which has appeared on the illicit drug market. The aim of this study was to identify the MOPPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and GC–MS. Analysis of urine samples of rats treated with MOPPP revealed that MOPPP [limit of detection (S/N 3) was 100 ng/ml] was completely metabolized by demethylation of the methoxy group, hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam and/or oxidative desamination to the corresponding diketo compounds. To some extent, the demethylated MOPPP metabolites were hydroxylated with partial subsequent methylation in position 3′. The hydroxy groups were found to be partly conjugated. Based on these data, MOPPP could be detected in urine via its metabolites by full-scan GC–MS using MS for screening and library search for identification by comparison of the spectra with reference spectra.
Keywords: 4′-Methoxy-α-pyrrolidinopropiophenone;

A capillary electrophoresis method for the enantiomeric separation of the tetrapeptide H–Tyr–(d)Ala–Phe–Phe–NH2 (TAPP), has been developed and validated. The preferred background electrolyte (BGE) consisted of 0.1 M aqueous phosphoric acid adjusted to pH 3.0 with triethanolamine. The chiral selectors 18-crown-6-tetracarboxylic acid (18C6H4) and heptakis(2,6-di-O-methyl)-β-cyclodextrin (2,6-DM-β-CD) were compared and the crown ether 18C6H4 was found to be superior. The separation of TAPP and its enantiomer was accomplished within 30 min with a resolution greater than 3.5. The method was then investigated with respect to selectivity, linearity, accuracy, range, precision, detection limit (DL), quantitation limit (QL) and robustness, essentially following International Conference of Harmonisation (ICH) guidelines for the validation of analytical methods. The DL and QL for the TAPP enantiomer were found to be 0.3 and 0.8%, respectively, at the target TAPP concentration of 1 mg/ml. Robustness was tested using a full factorial design for the following four experimental variables varied at two levels: pH of the BGE, chiral selector concentration in the BGE, phosphoric acid concentration in the BGE, and temperature. The method showed good performance with respect to all of the validation parameters, and proved to be robust to changes in the experimental parameters within the tested domain.
Keywords: 18-Crown-6-tetracarboxylic acid; H–Tyr–(d)Ala–Phe–Phe–NH2; Peptides;

This method is the first analytical method for the detection and quantitation of carfentanil and naltrexone at clinically relevant concentrations using liquid chromatography–mass spectrometry. Samples were alkalinized with 100 μl of 1 M NaOH and extracted 2× with 2 ml of toluene. The extractions were combined and dried under N2 at 40 °C in a H2O bath. Chromatography was performed using a Zirchrom PBD column and a mobile phase of 30:70 acetonitrile/10 mM ammonium acetate and 0.1 mM citrate (pH=4.4) at a flow rate of 0.3 ml/min. The lower limit of quantitation was 8.5 pg/ml for carfentanil and 0.21 ng/ml for naltrexone.
Keywords: Carfentanil; Naltrexone;

Cytochrome P450 3A4 activity displays a wide variability. The urinary 6β-hydroxycortisol to cortisol ratio, as a non-invasive assay, can be useful for its pretherapeutic characterization. We developed an HPLC–UV method preceded by liquid–liquid extraction for assessment of this ratio in clinical practice. Urine was collected on second void morning-spot sample. Percentage recoveries were high and reproducible. The 6β-hydroxycortisol to cortisol ratio ranged from 1.6 to 9.9 in 12 Caucasian healthy volunteers. It was reduced by 30 to 70% after ingestion of white grapefruit juice, a CYP3A4 inhibitor. Our method, simple, sensitive and accurate, could be helpful for determination of CYP 3A4 activity before oral chemotherapy, or for the monitoring of the use of grapefruit juice as a pharmacological modulator.
Keywords: Cortisol; 6β-Hydroxycortisol;

A validated method was developed for the simultaneous determination of the hydroxycinnamates caffeic (CA), dihydrocaffeic (DHCA), ferulic (FA), dihydroferulic (DHFA), and isoferulic acid (IFA) and the flavonoid luteolin (LUT) in human plasma as metabolites derived from artichoke leaf extract. The method involves sample preparation followed by separation using high-performance liquid chromatography on reversed-phase material with a polar endcapping (Aqua-C18, 250×4.6 mm). Selectivity and sensitivity towards the target compounds were achieved by electrochemical array detection (CoulArray). Calibration curves were constructed in the ranges 2.1–51.7 ng mL−1 (CA), 2.0–76.7 ng mL−1 (DHCA), 2.2–53.7 ng mL−1 (FA), 2.1–79.2 ng mL−1 (DHFA), 1.1–52.6 ng mL−1 (IFA) and 2.1–258.6 ng mL−1 (LUT). Linearity could be shown for all target compounds over the entire calibration range. Values for within-day and between-day precision and accuracy were in accordance with the international guidelines for validation of bioanalytical methods. It is concluded that this newly developed method is appropriate for analysing samples from bioavailability and pharmacokinetic studies after oral administration of artichoke leaf extract.
Keywords: Hydroxycinnamates;

R,S-3′,4′-Methylenedioxy-α-pyrrolidinopropiophenone (MDPPP) is a new designer drug with assumed amphetamine-like effects, which has appeared on the illicit drug market. The aim of this study was to identify the MDPPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and GC–MS. Analysis of urine samples of rats treated with MDPPP revealed that MDPPP was completely metabolized by demethylenation of the methylenedioxy group followed by partial 3′-methylation of the resulting catechol, oxidative desamination to the corresponding diketo compounds and/or hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam. The hydroxy groups were found to be partly conjugated. Based on these data, MDPPP could be detected in urine via its metabolites by full-scan GC–MS using mass chromatography for screening and library search for identification by comparison of the spectra with reference spectra.
Keywords: 3′,4′-Methylenedioxy-α-pyrrolidinopropiophenone;

Enantioselective separation methods and the enantioselective determination of the anti-allergic drug azelastine and of three of its main phase I metabolites in a biological matrix underwent chromatographic and electrophoretic investigations. An enantioselective assay of a coupling of HPLC using a β-cyclodextrin chiral stationary phase to ionspray tandem mass spectrometry is presented. Additionally, this assay is compared to another enantioselective assay using electrokinetic capillary chromatography with β-cyclodextrin and carboxymethyl-β-cyclodextrin in polyacrylamide-coated capillaries. For capillary electrophoresis (CE) the importance of polyacrylamide coating for the validation of this separation method is highlighted. Extracted rat plasma samples of enantioselective metabolism studies were measured by both validated assays. Differences in the pharmacokinetics and pharmacodynamics were evaluated for the main substance azelastine and its main metabolite demethylazelastine. So, a first hint about the enantioselectivity of biotransformation of azelastine in rats was seen after oral application of either enantiomer or the racemate to rats.
Keywords: Azelastine; Demethylazelastine;

Relative mRNA expression of the lactate dehydrogenase A and B subunits as determined by simultaneous amplification and single strand conformation polymorphism by Ze-Jun Liu; Wei-Cong Peng; Xin Yang; Jun-Fu Huang; Xiao-Bing Zhang; Yun Zhang; Masato Maekawa (405-412).
To explore if it is correlated in human tumor cells that the expression of LDH homologous gene and LDH isoenzymes, we used RT-PCR-SSCP technique to measure the relative expression of genes with homologous sequences. The combination of PCR using common primers designed in the highly conserved regions and single-strand conformation polymorphism analysis of the products is used for quantitative determination of the proportions of LDH-A mRNA in human cancer cell lines. The proportion is compared with that of the activities of isoenzymes. The results indicated that the enzyme activity of LDH-A was consistent with mRNA levels in the human tumor cell. The present procedure using a single pair of primers for two fragments can overcome disadvantages in quantitative analysis using multiplex PCR. Template concentrations and PCR cycles did not affect the proportions of LDH-A and LDH-B in the product.
Keywords: Enzymes; Lactate dehydrogenase; Homologous gene;

A new simple and specific method was developed and validated for the quantitative determination of OSI-774 (Tarceva™, Erlotinib) and its metabolite, OSI-420, in human plasma. Sample pretreatment involved a single protein precipitation step with acetonitrile. The analytes were separated on Waters X-Terra C18 (50×2.1 mm I.D., 3.5 μm) analytical column and eluted with acetonitrile–water mobile (70:30, v/v) containing 0.1% formic acid. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The overall extraction efficiency was greater than 88% for OSI-774 and 62% for OSI-420, with values for within-day and between-day precision and accuracy of <15%. Compared to previous assays, this method is simple, specific, and reproducible and will be used to characterize the plasma pharmacokinetics of OSI-774 at doses of 50 to 150 mg to optimize treatment with this agent.
Keywords: OSI-774; OSI-420;

A simple and sensitive high-performance liquid chromatographic (HPLC) method using ultraviolet detection was developed for the determination of testosterone in human plasma. Testosterone and the internal standard, griseofulvin, were extracted from 0.50 ml plasma sample using a mixture of dichloromethane–2,2,4-trimethylpentane (3:2, v/v). The mobile phase, consisted of 0.02 M sodium dihydrogenphosphate–acetonitrile–methanol (51:47:2, v/v) adjusted to pH 3.1 and delivered to a C18 analytical column (150×4.6 mm I.D., 4 μm particles) at a flow-rate of 1 ml/min while the detection wavelength was set at 240 nm with a sensitivity range of 0.005 a.u.f.s. The method has a quantification limit of 1.6 ng/ml. Recoveries of testosterone were all greater than 92% over the linear concentration range of 1.6–400 ng/ml while that of griseofulvin was approximately 95%. The within- and between-day RSD values were all less than 8% while the accuracy values ranged from 96.0 to 106.0% over the concentration range studied. The method was applied to the analysis of early morning plasma testosterone levels of 12 healthy human male volunteers. The levels were found to range from 3.1 to 8.4 ng/ml, within the normal range reported in the literature.
Keywords: Testosterone;