Biomedical Chromatography

López‐Cervantes, J.; Sánchez‐Machado, D. I.; Gutiérrez‐Coronado, M. A.; Ríos‐Vázquez, N. J.

doi: 10.1002/bmc.676pmid: 16802328

In the present study, a simple and rapid reversed‐phase HPLC method for the determination of astaxanthin in shrimp waste hydrolysate has been developed and validated. The analytical procedure involves the direct extraction of astaxanthin from the lipid fraction with methanol. The analytical column, SS Exil ODS, was operated at 25C. The mobile phase consisted of a mixture of water:methanol:dichloromethane:acetonitrile (4.5:28:22:45.5 v/v/v/v) at a flow rate of 1.0 mL/min. Detection and identification were performed using a photodiode array detector (λdetection = 476 nm). The proposed HPLC method showed adequate linearity, repeatability and accuracy. Copyright © 2006 John Wiley & Sons, Ltd.

Cerkvenik‐Flajs, Vesna

doi: 10.1002/bmc.599pmid: 16521165

Validation of an analytical method for determining chloramphenicol residues in muscle tissue by gas chromatography–electron capture detection (GC‐ECD) was performed according to the latest European Union criteria for the analysis of veterinary drugs in food, laid down by Commission Decision 2002/657/EC. The method using the meta isomer of chloramphenicol as an internal standard proved to be very selective, specific to other related phenicols and accurate to within +3.6% at a concentration level of 8.9 µg/kg, as present in the certified reference material available. The correlation coefficient of the calibration curve was 0.9991. At all three fortification levels studied (0.3, 0.45 and 0.6 µg/kg), repeatability and intra‐laboratory reproducibility were <8 and ≤9%, respectively. The decision limit (CCα) and detection capability (CCβ) were 0.07 and 0.12 µg/kg, respectively. The validation results and the results of participation in an international inter‐laboratory proficiency test indicate that the method presented is completely suited for regulatory control to screen and quantify chloramphenicol residues in various muscle tissues on a routine basis. Copyright © 2006 John Wiley & Sons, Ltd.

Shi, Yunfeng; He, Langchong; Wang, Sicen

doi: 10.1002/bmc.614pmid: 16583458

A gas chromatography/mass spectrometry (GC/MS) method was developed to study the pharmacokinetics of ligustilide following oral administration to rats. The method was used for the analysis of samples taken from rats. Biological samples were prepared by liquid–liquid extraction (LLE) using an n‐hexane–ether (2:1) solvent mixture for a sample clean‐up step and analyzed by GC/MS with a quadrupole MS detector in selected ion monitoring mode (m/z 190). The calibration curves were linear over the concentration range 0.172–8.60 µg/mL (r > 0.99) for blood samples and a different range (r > 0.99) for different tissue samples. The limit of detection (LOD) was 1.0 ng/mL or 1.0 ng/g (three times the signal–noise ratio). Within‐ and between‐day precision expressed as the relative standard deviation (RSD) for the method was 1.58–3.88 and 2.99–4.91%, respectively. The recovery for all samples was >80%, except for liver samples (>70%). The main pharmacokinetic parameters obtained were: Tmax = 0.65 ± 0.07 h, Cmax = 1.5 ± 0.2 µg/mL, AUC = 34 ± 6 h µg/mL and Ka = 3.5 ± 0.6/h. The experimental results showed that ligustilide was easily absorbed, but its elimination was slow, from 3 to 12 h after oral administration. The concentrations of ligustilide in rat cerebellum, cerebrum, spleen and kidney were higher than those in other organs. Copyright © 2006 John Wiley & Sons, Ltd.

Ryu, Hye Kyung; Jung, Byung Hwa; Kim, Kyung Mee; Yoo, Eun Ah; Woo, Jeong‐Taek; Chung, Bong Chul

doi: 10.1002/bmc.615pmid: 16544269

This work describes a sensitive method for determining cholesterol in human hair using GC‐MS. In this study, we used a very small amount of hair, only 1 mg, to quantify cholesterol. We also can achieve more effective purification and a good recovery over 92% with solid‐phase extraction using an Oasis HLB cartridge. The intra‐day and inter‐day precision and accuracy values were less than 7.08%. Cholesterol was determined to be in the range of 355–1693 µg/g in healthy human hair. We tested the concentration correlation between the serum and hair to examine the feasibility of using the hair cholesterol level as an index of the serum cholesterol level. The correlation between the serum cholesterol was 0.86 (r‐value) in patients with hypercholesterolemia. This finding indicates that, in the clinical field, hair could replace serum in cholesterol level measurement. Copyright © 2006 John Wiley & Sons, Ltd.

Brzezińska, Elżbieta; Kośka, Grażyna

doi: 10.1002/bmc.621pmid: 16506293

A structure–activity relationship (SAR) analysis of H1‐, H2‐ and H3‐antihistamine activity was carried out and chromatographic data of 2‐(2‐(phenylamino)thiazol‐4‐yl)ethanamine, 2‐(2‐benzyl‐4‐thiazolyl)ethanamine, 2‐(2‐benzhydrylthiazol4‐yl)ethanamine, 2‐(1‐piperazinyl‐ and 2‐(hexahydro‐1H‐1,4‐diazepin‐1‐yl)benzothiazole, 2‐(1‐piperazinyl)benzothiazole, 2‐(4‐(1‐alkyl)piperidinyl)benzothiazole, 2‐(N,N′,N′‐dimethylalkyl‐1,2‐ethanediamino)benzothiazole, 2(1‐(4‐aminopiperidinyl))benzothiazole, 2‐(2‐phenyl‐4‐thiazolyl)ethanamine derivatives and selected H1‐ and H2‐antihistamine drugs were obtained. NP TLC and RP2 TLC plates (silica gel NP 60F254 and silica gel RP2 60F254 silanized precoated), impregnated with a solution of aspartic acid (l‐Asp) and a solution of an analogue of aspartic acid (propionic acid), were used in two developing solvents as H1‐, H2‐ and H3‐antihistaminic interaction models. The lipophilicity data of the examined compounds were obtained and used in the SAR assay. Biochromatographic tests using TLC plates impregnated with solutions of asparic acid or propionic acid were found to be a source of useful data for the qualitative analysis of compounds with different structures, demonstrating activity to histamine H1‐, H2‐ and H3‐receptors. The four presented discriminant models based on biochromatographic studies are an efficient tool in the SAR analysis for initial prediction of compound activity direction within histamine receptors. Copyright © 2006 John Wiley & Sons, Ltd.

Yao, Ming; Srinivas, Nuggehally

doi: 10.1002/bmc.627pmid: 16506266

A high‐performance liquid chromatographic–mass spectrometric (LC/MS) assay was developed and validated for the determination of muraglitazar, a novel α/γ, dual PPAR activator, in monkey plasma. The method utilized trazodone as the internal standard (IS). The extraction scheme involved a simple protein precipitation procedure with the use of a mixture of acetonitrile and methylene chloride. Separation was carried out on a BDS Hypersil C18 analytical column (2 × 50 mm, 3 µm) and an effective chromatographic separation of muraglitazar (3.31 min) and trazadone (2.27 min) was achieved at a ßow rate of 0.3 mL/min. The mobile phase, used in an isocratic mode, consisted of 90% A (acetonitrile: 0.1% formic acid, 50:50 v/v) and 10% B (acetonitrile: 0.1% formic acid, 95:5 v/v). Detection of muraglitazar and trazodone was by positive ion turbo‐ion spray mass spectrometry in the SIM mode. The mass spectrometer was programmed to admit the protonated molecules at m/z 372.0 (IS) and m/z 517.1 (muraglitazar). The standard curve, which ranged from 2 to 500 ng/mL, was fitted to a 1/x weighted linear regression model. The between run precision and within‐run precision values of the assay was within 6.2% RSD. The assay accuracy was within 10.0% of the nominal values of the range of QC samples (6.0–400 ng/mL). At the lower limit of quantitation (LLQ) of 2 ng/mL, the deviation of the predicted concentrations from the nominal value of LLQ samples (n = 6) were within ±16.6%. Muraglitazar was stable in monkey K3EDTA plasma for at least three freeze–thaw cycles. The processed samples (spiked samples) were stable for 48 h in auto‐sampler at 10°C. The average extraction recoveries of muraglitazar and IS were 83.3 and 91.9%, respectively. The assay was applied to delineate the pharmacokinetic disposition of muraglitazar in monkeys following a single oral dose. Copyright © 2006 John Wiley & Sons, Ltd.

García, Javier; Márquez, Apolonia; Ruiz, Rosa; López, Luis F.; Claro, Carmen; Lucero, María Jesús

doi: 10.1002/bmc.629pmid: 16583452

An isocratic high‐performance liquid chromatographic method with UV detection has been developed and validated for the quantitative determination of foscarnet in isoosmotic sodium chloride aqueous solution. The mobile phase consisted of mixture of methanol:water (30:70 v/v), containing 1 mm tetrahexylammonium hydrogen sulphate at pH 5.80. The analyte was separated on a reversed‐phase C18 column packed with 4 µm spherical particles of octadecylsilane. Hydrochlorothiazide was used as internal standard. UV detection at 232 nm allowed a quantification limit of 50 µg/mL. The assay was linear from 50 to 4000 µg/mL. The coefficient of variation was ≤2.52% for intra‐assay precision and ≤3.49% for inter‐assay precision. The deviation from the nominal value ranged from −0.57 to 0.47% for the same‐day accuracy and from −0.75 to 3.06% for day‐to‐day accuracy. Copyright © 2006 John Wiley & Sons, Ltd.

Mudigonda, Koteshwara; Jukanti, Raju; Apte, Shashank S.; Ajjala, Devender R.; Shrivastava, Wishu; Kandikere, Vishwottam N.; Nirogi, Ramakrishna V. S.

doi: 10.1002/bmc.631pmid: 16506264

A rapid, reliable HPLC method with UV detection (240 nm) was developed and validated for quantitation of saquinavir in mice brain and testis. Saquinavir and the internal standard were isolated from homogenized tissue matrices using liquid–liquid extraction procedure and were then analyzed using an isocratic mobile phase by reversed‐phase liquid chromatography. The lower limit of quantification was 50 ng/g for both brain and testis. A linear dynamic range of 50–5000 ng/g for both brain and testis was established. This HPLC method was validated with between‐batch precision of 0.5–4.4 and 1.5–5.5% for brain and testis, respectively. The between‐batch accuracy was 94.7–105.9% and 97.5–105.0% for brain and testis, respectively. The present method was applied for tissue distribution studies of the novel drug delivery systems of saquinavir in mice. Copyright © 2006 John Wiley & Sons, Ltd.

Cheremina, Olga; Brune, Kay; Hinz, Burkhard

doi: 10.1002/bmc.633pmid: 16602135

Lumiracoxib {2‐((2‐fluoro‐6‐chlorophenyl)amino)‐5‐methyl‐benzeneacetic acid} is a highly selective and potent cyclooxygenase‐2 (COX‐2) inhibitor, which is chemically distinct from other coxibs in that it contains a carboxylic group and is weakly acidic. In the present study, a liquid–liquid extraction‐based reversed‐phase HPLC method with UV detection was validated and applied for the analysis of lumiracoxib in human plasma. The analyte was separated on a reversed‐phase column with acetonitrile and 0.05% trichloracetic acid in water (35:65, v/v) as mobile phase at a flow rate of 1 mL/min, and UV detection at 270 nm. The retention times for lumiracoxib and niflumic acid (internal standard) were 16.9 and 10.4 min, respectively. The validated quantitation range for lumiracoxib was 10–10,000 ng/mL. The developed procedure was applied to assess the pharmacokinetics of lumiracoxib following administration of a single oral 200 mg dose to a healthy male volunteer. Copyright © 2006 John Wiley & Sons, Ltd.

Cai, Hong; Raynaud, Delphine; Steward, William P.; Gescher, Andreas J.

doi: 10.1002/bmc.634pmid: 16506281

The flavone apigenin occurs in many leafy vegetables and fruits. It has been reported to have cancer chemopreventive efficacy in rodents. An HPLC method described previously for the determination of tricin, the dimethoxy cogener of apigenin, was modified and validated for measurement of apigenin in mouse tissues. Separation was carried out on a Hypersil‐BDS C18 column (4.6 × 250 mm) with an isocratic mobile phase of 55% methanol in 0.1 m ammonium acetate, pH 5.10, containing 0.27 mm disodium ethylenediamine tetraacetic acid. UV detection was at 336 nm, without interference from endogenous tissue compounds. The assay was linear in the range 25–400 ng/mL, 0.25–4 µg/mL and 2.5–40 µg/mL, with r2 > 0.99 in all cases, for mouse plasma, liver and intestinal mucosa, respectively. Apigenin in mouse plasma, liver and intestinal mucosa was efficiently extracted with 0.1 m acetic acid in acetone. The assay recovery at low, medium and high concentrations was between 94.6 and 131.7% for all biomatrices, with a relative standard deviation of <10%. The lower limit of quantification for plasma was 25 ng/mL with a relative standard deviation of <15%. The method was used to measure the steady‐phase apigenin levels in tissues of mice receiving apigenin in their diet. Copyright © 2006 John Wiley & Sons, Ltd.