Biomedical Chromatography

Gallardo, E.; Queiroz, J. A.

doi: 10.1002/bmc.1009pmid: 18506679

The use of alternative specimens in the field of toxicology was first described in 1979, when hair analysis was used to document chronic drug exposure. Since then, the use of these ‘alternative’ samples has gained tremendous importance in forensic toxicology, as well as in clinic toxicology, doping control and workplace drug testing. It is not surprising, therefore, that a large number of papers dealing with the determination of several classes of drugs in saliva, sweat, meconium and hair have been published ever since, owing to the fact that chromatographic equipment is becoming more and more sensitive, mass spectrometry (and tandem mass spectrometry) being the most widely used analytical tool, combined with gas or liquid chromatography. ‘Alternative’ specimens present a number of advantages over the ‘traditional’ samples normally used in toxicology (e.g. blood, urine and tissues), namely the fact that their collection is not invasive, their adulteration is difficult, and they may allow increased windows of detection for certain drugs. The main disadvantage of this kind of samples is that drugs are present in very low concentrations, and therefore high‐sensitivity techniques are required to accomplish the analysis. This paper reviews a series of publications on the use of alternative specimens, with special focus on the main analytical and chromatographic problems that these samples present, as well on their advantages and disadvantages over traditional samples in documenting drug exposure. Copyright © 2008 John Wiley & Sons, Ltd.

Park, Jin‐Hee; Park, Yoo‐Sin; Lee, Min‐Ho; Rhim, Si‐Youn; Song, Jae‐Chul; Lee, Soo‐Jin; Kim, Jung‐Mogg; Shaw, Leslie M; Kang, Ju‐Seop

doi: 10.1002/bmc.995pmid: 18348335

A rapid, simple and validated liquid chromatography coupled to tandem mass spectrometric method (LC‐MS/MS) for topiramate analysis in human plasma has been applied to pharmacokinetic and bioequivalence studies in 24 healthy male Korean volunteers. The procedure involves a simple liquid extraction of topiramate and prednisone (internal standard) with acetonitrile and separation by HPLC equipped with a Capcell Pak C18 column using acetonitrile–0.1% triethylamine (80:20, v/v) as a mobile phase. Detection was carried out on an API 2000 MS system by multiple reactions monitoring mode. The ionization was optimized using ESI(−) and selectivity was achieved by MS/MS analysis, m/z 338.0 → 77.5 and m/z 357.1 → 327.2 for topiramate and prednisone, respectively. The method had a total run time of 2.5 min and showed good linearity over a working range of 20–5000 ng/mL in human plasma with a lower limit of quantification of 20 ng/mL. No metabolic compounds were found to interfere with the analysis. The inter‐day and intra‐day accuracy were in the ranges of 99.24–116.63 and 93.45–108.68%, respectively, and inter‐day and intra‐day precisions were below 6.24 and 5.25%, respectively. This method was successfully applied for pharmacokinetic and bioequivalence studies by analysis of blood samples taken up to 96 h after an oral administration of 100 mg of topiramate in 24 healthy Korean volunteers. Copyright © 2008 John Wiley & Sons, Ltd.

Banerjee, Suchitra; Madhusudanan, K. P.; Chattopadhyay, Sunil K.; Rahman, Laiq Ur; Khanuja, Suman P. S.

doi: 10.1002/bmc.998pmid: 18386250

Agrobacterium rhizogenes‐mediated ‘hairy root’ cultures were established in Atropa acuminata. The chemical profiling of the hairy roots was carried out by a new mass spectrometric technique, direct analysis in real time (DART). The intact hairy roots were directly analyzed by holding them in the gap between the DART ion source and mass spectrometer. Two alkaloids, atropine and scopolamine, were characterized. The structural confirmation of the two alkaloids was made through their accurate molecular formula determinations. This is the first report of establishing hairy roots in A. acuminata as well as application of the DART technique for the chemical profiling of its hairy roots. Copyright © 2008 John Wiley & Sons, Ltd.

Ma, Chunhui; Fan, Mingsong; Tang, Yihong; Li, Zhixiong; Sun, Zhaolin; Ye, Guan; Huang, Chenggang

doi: 10.1002/bmc.1000pmid: 18318017

Huangbai‐Zhimu herb‐pair (HBZMHP) is a widely used Chinese traditional medicine formula in treating various diseases; however, its active components have remained unknown. In this paper, serum chemistry and combined high‐performance liquid chromatography (HPLC), diode‐array detection and mass‐spectrometry (MS) techniques were used to study the constituents of HBZMHP extract absorbed into rat serum after oral administration. A total of nine characteristic HPLC peaks in the TIC chromatograms were identified as magnoflorine (1), menisperine (2), palmatine (3), berberine (4), timosaponin N or timosaponin E1 (5), timosaponin D (6), timosaponin BIII, anemarsaponin C or xilingsaponin B (7) timosaponin BII (8) and timosaponin AIII (9). All of the identified peaks were constituents of HBZMHP extract. The results narrow the range of active compounds to be found in HBZMHP extract, and pave the way for the follow‐up action mechanism research. Copyright © 2008 John Wiley & Sons, Ltd.

Tang, Wen‐Fu; Wan, Mei‐Hua; Huang, Qiao‐Rong; Zhu, Zheng‐Yan; Zhao, Jiang‐Lei; Wu, Yi‐Zhi; Huang, Xi

doi: 10.1002/bmc.1002pmid: 18384065

Da Cheng Qi decoction (DCQD) is composed of Dahuang, Houpu, Zhishi and Mangxiao. It is a formula created under the theory of Chinese medicine to purge the ‘evil heat’ in the gastrointestitinal tract, which arises from the ileus and acute pancreatitis. The present study was conducted to evaluate the herb–drug interaction between DCQD and ranitidine, which are often co‐administered in clinical practice. Ranitidine was administered orally alone or together with DCQD to rats, and plasma ranitidine concentrations were measured by HPLC. Following oral administration, ranitidine plasma levels revealed curves characterized by peaks at 1.8 and 4.2 h corresponding to ranitidine alone and ranitidine with DCQD at mean concentrations of 16.315 and 1.455 µg/mL, respectively. After ranitidine was orally dosed alone or with DCQD, the half‐lives were 1.787 and 3.758 h, while the area under the concentration–time curve (0–12 h) was 28.083 and 9.826 µg/L h, respectively, suggesting that DCQD might significantly affect the pharmacokinetics of ranitidine in rats. When physicians or pharmacists administer DCQD and ranitidine, they must make a careful effort to adjust the dosage of the drug and Chinese decoction, or avoid the herb–drug co‐administration. Copyright © 2008 John Wiley & Sons, Ltd.

Goda, Ryoya; Masumoto, Hiroshi; Okazaki, Osamu; Sudo, Kenichi

doi: 10.1002/bmc.1003pmid: 18348334

Compared with a standard gradient system, the new gradient system which we developed has a major advantage because it permits a wide range of acetonitrile content, e.g. more than the critical threshold, in the polypeptide solution and allows the quantitative analysis of the polypeptide with satisfactory analytical precision. Additionally, this new gradient system allows the enhancement of the sensitivity of the polypeptide analysis proportionate to the increased volume of solution loaded with the same levels of precision. In contrast, when using a standard gradient system it is difficult to analyze a polypeptide quantitatively with good precision due to either adsorption to various materials or to irregular change in the ratio between a retained and a passed peak of the polypeptide. Additionally, the appearance of a passed peak results in a loss in the sensitivity of the polypeptide analysis, although no adsorption of a polypeptide to various materials occurs in a solution with acetonitrile content more than the critical threshold. Consequently, the new gradient system is effective for the simultaneous and quantitative analysis of different polypeptides with good precision and without any loss of sensitivity due to either adsorption to various materials or the appearance of a passed peak. Copyright © 2008 John Wiley & Sons, Ltd.

Li, Li‐bo; Zhang, Jin‐lan; Wang, Yu‐xiang; Wei, Huai‐ling; Liu, Geng‐tao

doi: 10.1002/bmc.1004pmid: 18384063

An HPLC method was established and validated for the determination of compound FLZ, a synthetic novel anti‐Parkinson's disease candidate drug, in rat plasma. FLZ and the internal standard bicyclol were extracted from plasma by solid‐phase extraction method and analyzed on a Restek C18 column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of methanol and water (60:40, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was set at 320 nm. The calibration curve was linear within the concentration range from 25 to 500 ng/mL (r2 > 0.999), the limit of quantitation was 25 ng/mL and the average recovery was 92.0% with the RSD less than 5.9%. The relative standard deviation for intra‐ and inter‐day precision was less than 3.8 and 6.9%, respectively. The established HPLC method was validated to be a simple, rapid and reliable procedure and applied to study the preclinical pharmacokinetics of FLZ in rat plasma, and it was the first time that the pharmacokinetics of FLZ had been investigated. Copyright © 2008 John Wiley & Sons, Ltd.

Zhang, Yan; Ding, Likun; Tian, Yun; Yang, Jing; Yang, Lin; Wen, Aidong

doi: 10.1002/bmc.1005pmid: 18318018

A sensitive and specific method was developed and validated for the determination of mitiglinide in human plasma using liquid chromatographic separation with electrospray ionization tandem mass spectrometric detection. Acidified plasma samples were extracted with ethyl acetate. The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C18 column with a mobile phase of methanol–10 mm ammonium acetate solution at a flow rate of 0.3 mL/min. Analytes were detected with an Agilent 6410 Triple qudrupole mass spectrometer equipped with an electrospray ionization source in positive multiple reaction monitoring mode: m/z 316.2 (precursor ion) to 298.2 (product ion) for mitiglinide and m/z 318.2 (precursor ion) to 120.2 (product ion) for the internal standard. This method was validated over a linear range of 0.5–4000 ng/mL for mitiglinide in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/mL, while a relative standard deviation (RSD) was less than 3.9%. The intra‐ and inter‐run precision (as RSD, %) obtained from three validation runs were all less than 15%. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies. Copyright © 2008 John Wiley & Sons, Ltd.

Zeng, L.; Nath, C. E.; Shaw, P. J.; Earl, J. W.; McLachlan, A. J.

doi: 10.1002/bmc.1006pmid: 18348336

A simple, accurate, reliable and sensitive HPLC method was developed and validated for quantitating acyclovir in human plasma. Sample (100 µL) preparation involved addition of guanosine (internal standard) and protein precipitation with 7% perchloric acid and centrifugation. Supernatant (20 µL) was injected onto a C18 HPLC column with a mobile phase of 0.05 m sodium phosphate buffer–acetonitrile (pH 2.35, 992:8, v/v) with 25 µL of 0.4 m tetrabutylammonium hydroxide titrant and fluorescence detection (excitation, 260 nm; emission, 375 nm). Analyte recovery was 101% and the assay response was linear over the acyclovir concentration range of 0.1–20 mg/L. Intra‐ and inter‐day accuracy and precision were less than 7%. The limit of detection and limit of quantitation were 0.033 and 0.1 mg/L, respectively. In five paediatric oncology patients administered intravenous acyclovir, concentrations ranged from 0.24 to 43.65 mg/L. This method can be used to measure acyclovir concentrations in paediatric patients. Copyright © 2008 John Wiley & Sons, Ltd.

Sasaki, Tsukasa; Fukushima, Takeshi; Ohishi, Madoka; Toyo'oka, Toshimasa

doi: 10.1002/bmc.1007pmid: 18318012

We developed a novel derivatization reagent, (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)‐(+)‐6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (α‐CA), which exhibits intense EC response. NPCA successfully yielded α‐CA derivatives of primary amines by a two‐step derivatization procedure. Following pre‐column derivatization with NPCA, a simultaneous determination of α‐CA derivatives of neuroactive monoamines (dopamine (DA), epinephrine, and 5‐hydroxytryptamine (5‐HT)), their monoamine oxidase metabolites (3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindole‐3‐acetic acid) and their catechol‐O‐methyltransferase metabolites (3‐methoxytyramine (3‐MT) and normetanephrine (NMN)) was completely achieved using our HPLC‐EC method. Using an HPLC equipped with coulometric electrode‐array detection system, the resultant α‐CA derivatives of NMN, 5‐HT, DA and 3‐MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4‐fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16–60 fmol on column (signal‐to‐noise ratio 3). The proposed HPLC method was applied to determine 5‐HIAA, HVA, α‐CA‐5‐HT and α‐CA‐DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions. Copyright © 2008 John Wiley & Sons, Ltd.