Showing papers in "Biomedical Chromatography in 2003"

Enantioseparation of some clinically used drugs by HPLC using cellulose Tris (3,5-dichlorophenylcarbamate) chiral stationary phase.

Abstract: The chiral resolution of some clinically used drugs namely metoprolol, teratolol, tolamolol, nebivolol (β-adrenergic blockers), econazole, miconazole (anti-fungal agents), cromakalim (anti-hypertensive agent) and etodolac (anti-inflammatory agent) was achieved on cellulose tris (3,5-dichlorophenylcarbamate) chiral stationary phase. The mobile phase used was 2-propanol at 0.5 mL/min with detection at 220 nm. The separation factors (α) of these drugs ranged from 1.24 to 3.90 while the resolution factors were from 1.05 to 5.0. The chiral recognition mechanisms between the racemates and the chiral selector are discussed. Copyright ­© 2003 John Wiley & Sons, Ltd.

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high‐performance liquid chromatography

Abstract: Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.

Quantification of polycyclic aromatic hydrocarbons (PAHs) in human hair by HPLC with fluorescence detection: a biological monitoring method to evaluate the exposure to PAHs.

Abstract: A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs) in human hair. Fifteen kinds of PAHs classified as priority pollutants by the US EPA were quantified with four perdeuterated PAHs as internal standards. After 50 mg hair samples were washed with n-hexane to remove external contamination of PAHs, the samples were digested in 2.5 M sodium hydroxide. The digests were extracted with n-hexane and then analyzed by HPLC. Eleven kinds of PAHs were identified in hair samples of 20 subjects, and 10 kinds of PAHs were eventually quantified using the internal standards. For anthracene, chrysene and benzo[k]fluoranthene, significant differences were observed between smokers and non-smokers. Although benzo[b]fluoranthene, dibenz[a,h]anthracene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were observed in the particulates of indoor and outdoor air, they were not detected in all hair samples. The analysis of PAHs in human hair should be useful as a new biomarker to evaluate the exposure to PAHs.

Lophine derivatives as versatile analytical tools.

Abstract: Lophine (2,4,5-triphenylimidazole) derivatives as versatile analytical tools in biomedical sciences are described. Chemiluminescence (CL) and fluorescence (FL) properties of the lophine derivatives are first demonstrated including the CL reaction mechanism, effects of substituents on CL yields, FL spectral behaviors, etc. Next, analytical applications to the determination of metal ions such as cobalt (II) and chromium (VI) are discussed. Finally, the application studies of lophine derivatives as CL and FL reagents for the determination of organic substances in biological materials are presented. Among the derivatives, 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole (HCPI) and 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) are studied, with their excellent properties as labeling reagents for fatty acids and amines and/or phenols, respectively, in high-performance liquid chromatography. The utility of boronic acid derivatives as CL enhancers is also discussed in this review.

Simultaneous determination of leflunomide and its active metabolite, A77 1726, in human plasma by high‐performance liquid chromatography

Abstract: The isoxazol derivative leflunomide [N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide] is an inhibitor of de novo pyrimidine synthesis used for the treatment of rheumatoid artrithis. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of leflunomide and its active metabolite, A77 1726, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile, water and formic acid (40/59.8/0.2, v/v), at a flow rate of 0.5 mL/min, and UV detection at 261 nm. The retention times for A77 1726, leflunomide and warfarin (internal standard) were 8.2, 16.2 and 12.2 min, respectively. The validated quantification range of the method was 0.05-100 micro g/mL for leflunomide and 0.1-100 micro g/mL for A77 1726. The developed procedure was applied to assess steady-state plasma concentrations of A77 1726 in patients with rheumatoid arthritis treated with 10 or 20 mg leflunomide per day.

Determination of paeonol in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies following oral administration of Moutan cortex decoction.

Abstract: Quantification of paeonol, the principal bioactive component of Moutan cortex, in rat plasma following oral administration of Moutan cortex decoction was achieved by using a simple and sensitive high-performance liquid chromatographic method. The calibration curves for paeonol were linear in both the low (25–200 ng/mL) and the high concentration range (200–4000 ng/mL) with r2 values of 0.9928 and 0.9993, respectively. The coefficients of variation of intra- and inter-day assays were 14.36, 6.52, 1.76, 1.25, 5.36, 3.30 and 1.42% and 12.70, 1.19, 2.98, 1.91, 1.75, 1.78 and 0.96% at concentrations of 25, 50, 100, 200, 500, 1000 and 2000 ng/mL, respectively. The recoveries of paeonol from rat plasma were found to be 101.9, 104.5, 105.4 and 101.2% for concentrations of 50, 500, 1000 and 2000 ng/mL, respectively. The paeonol plasma concentrations were fitted to two-compartment model with first order absorption. The mean terminal half-lives (t1/2) of paeonol was 80.9 min. Copyright © 2003 John Wiley & Sons, Ltd.

Biopartitioning micellar chromatography to predict skin permeability.

Abstract: Dermal absorption of chemicals is an area of increasing interest to the pharmaceutical and cosmetic industries, as well as in dermal exposure and risk assessment processes. In this paper the capability of biopartitioning micellar chromatography (BMC) as an in vitro technique to describe compound percutaneous absorption is evaluated. A multivariate study (principal component analysis, partial least squares) is performed in order to evaluate the importance of some physicochemical variables on the skin permeability constant values. From these results, a quantitative retention-activity relationship model for predicting the skin permeability constants that uses the BMC retention data and melting point as descriptor variables was obtained from a heterogeneous set of 43 compounds. The main advantage of the proposed methodology is that it allows the obtention of permeability constants of ionic compound and it can be very useful to predict the effect of pH of vehicle on the skin permeability of xenobiotics.

A simple and simultaneous determination of acyclovir and ganciclovir in human plasma by high-performance liquid chromatography.

Abstract: A simple high-performance liquid chromatographic method was developed for the simultaneous determination of the therapeutic levels of acyclovir and ganciclovir in human plasma. After precipitation of plasma proteins with 6% perchloric acid, acyclovir and ganciclovir were simultaneously determined by reversed-phase chromatography with spectophotometric detection at 254 nm. The peak heights for acyclovir and ganciclovir were linearly related to their concentrations ranging from 0.063 to 2.080 micro g/mL. The recovery was 100.48-102.84% for acyclovir and 99.26-103.07% for ganciclovir. The intra- and inter-day relative standard deviation values were in the range 0.186-8.703% for acyclovir and 0.137-6.424% for ganciclovir. The detection limits for both compounds were 0.01 micro g/mL determined as the signal-to-noise ratio of 3. The present method is applicable to therapeutic monitoring during antiviral medication.

Direct enantiomeric resolution of (+/-)-atenolol, (+/-)-metoprolol, and (+/-)-propranolol by impregnated TLC using L-aspartic acid as chiral selector.

Abstract: Resolution of three commonly used β-blockers, (±)-atenolol, (±)-metoprolol and (±)-propranolol, into their enantiomers has been achieved using normal-phase TLC on silica gel plates impregnated with L-aspartic acid as the chiral selector. Different combinations of acetonitrile–methanol–water as mobile phase were found to be successful in resolving the enantiomers. The spots were detected with iodine and the detection limits were found to be 0.26 µg for atenolol and 0.23 µg for each of metoprolol and propranolol as racemate. Copyright © 2003 John Wiley & Sons, Ltd.

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection.

Abstract: A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.

HPLC determination of ferulic acid in rat plasma after oral administration of Rhizoma Chuanxiong and its compound preparation.

Abstract: An HPLC method is described for determination of ferulic acid in rat plasma. The concentration of ferulic acid in rat plasma was determined after deproteinization with acetonitrile using sulfamethoxazole as internal standard. Chromatographic separations were performed on a C(18) stationary phase with a mobile phase composed of acetonitrile-water (16:84, v/v) with 1% glacial acetic acid. The UV detection wavelength was set at 320 nm. The method was successfully applied to the determination of pharmacokinetic parameters in rat plasma after oral administration of Rhizoma Chuanxiong and and its compound preparation Suanzaoren decoctions. The calibration curve was linear over the range 0.0510-4.08 micro g/mL in rat plasma. Within-day and between-day precisions were less than 4.5% RSD. Mean recovery was determined as 96.9%. The limit of quantitation was 0.0510 micro g/mL. The pharmacokinetic parameters of the two preparations were different significantly (p < 0.05), which may attribute to the effects of other ingredients present in Suanzaoren decoction.

Plasticizers, antioxidants, and other contaminants found in air delivered by PVC tubing used in respiratory therapy.

Abstract: Of the many compounds that leach from respiratory therapy tubing into air passing through it, we selected five compounds to analyze. The five compounds are known to be potentially carcinogenic, toxic or known to induce estrogenic activity. Parts-per-million and parts-per-billion concentrations of these species were found in the air passing through the tubing: the plasticizers di-(2-ethylhexyl) phthalate (DEHP) and di-ethyl phthalate (DEP), the antioxidants butylated hydroxy toluene (BHT) and p-nonylphenol (p-NP), and the contaminant (from commercial preparation of DEHP) 2-ethylhexanol (2-EH). These levels are high enough to cause some concern about exposure for patients who use oxygen on a long-term basis, those sensitive or allergic to these species, or those with asthma. A method was developed for analysis of solid tubing samples, showing great variability in concentrations of small, volatile molecules from sample to sample. A method was also developed for pre-concentration of small molecules onto Tenax adsorbants from air passing through the tubing. Both solid samples and adsorbant loaded with analyte were analyzed by direct dynamic thermal desorption gas chromatography mass spectrometry (GCMS). This study does not imply that adverse reactions by patients to chemical compounds leaching from respiratory medical tubing will occur but that further investigation is warranted.

Chemiluminescence detection in HPLC and CE for pharmaceutical and biomedical analysis.

Abstract: The present paper reviews the developments and applications of chemiluminescence detection with HPLC and CE in pharmaceutical and biomedical analysis. The chemiluminescence systems, chemiluminogenic reagents and derivatization reagents, improvements in instrumental design as well as their contributions to the practical applications, are all presented. The advantages and limitations of current detection methodology and future prospects for improvement are briefly discussed.

Determination of methamphetamine and amphetamine in abusers' plasma and hair samples with HPLC-FL

Abstract: This paper describes highly sensitive HPLC methods for the determination of amphetamine (AP) and methamphetamine (MP) in abusers' plasma and hair samples. AP and MP were derivatized with the fluorescent reagent, DIB-Cl, to yield a highly fluorescent DIB-derivatives of AP and MP, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 and 430 nm, respectively. The separation was achieved on an ODS column with isocratic mobile phases composed of acetoniltrile and citrate buffer (55:45, v/v) for plasma samples and of acetonitrile-methanol-citrate buffer (45:20:37.5, v/v/v) for hair samples. The limits of detection were less than 0.87 ng/mL and 0.12 ng/mg in plasma and hair samples, respectively, for both AP and MP. The methods were then applied to the determination of MP and its metabolite AP in plasma obtained from two cases of illegally ingested MP and in one of the cases' hair received later. Case I was treated with dialysis; samples before and after dialysis were analyzed by the described method. After dialysis for 5 h, the total plasma levels of AP and MP decreased from 720 to 190 ng/mL. For case II, MP and AP levels were monitored for 3 days after digestion. Total plasma levels decreased from 57 ng/mL in the day of digestion to 11 ng/mL after 3 days. In hair samples, AP and MP could also be detected in very low concentrations.

Analysis of intracellular regulatory proteins by immunoaffinity capillary electrophoresis coupled with laser-induced fluorescence detection.

Abstract: Measurement of intracellular regulatory proteins is of great importance in many areas of biomedical research. In this communication we describe an antibody-based capillary electrophoresis system equipped with an on-line laser-induced fluorescence detector capable of measuring intracellular proteins in cultures as low as 100 cells. The system demonstrated a high degree of precision and accuracy, being capable of detecting the fluorochrome-labeled analytes of interest at concentration of approximately 0.5 pg. We have used this instrument to study concentrations of the intracellular regulatory proteins STAT-1 and STAT-3, following stimulation of lymphocyte cultures with the inflammatory cytokine, IL-6. Using a combination of four antibodies specific for either STAT-1 or STAT-3 in both their nonphosphorylated and phosphorylated forms, we were able to demonstrate the differential expression of these proteins over time. Copyright © 2003 John Wiley & Sons, Ltd.

Reversed-phase liquid chromatographic method for the analysis of aminoglycoside antibiotics using pre-column derivatization with phenylisocyanate

Abstract: A pre-column derivatization liquid chromatographic method has been developed for the analysis of aminoglycoside antibiotics using phenylisocyanate as a derivatization reagent. Derivatives including kanamycin, neomycin and gentamicin were formed by reaction of the analytes with phenylisocyanate in the presence of triethylamine. Phenylisocyanato groups were attached to corresponding amino groups of aminoglycoside and their molecular mass was confirmed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The experimental conditions for derivatization and separation of aminoglycoside derivatives were optimized and validated. A simple liquid chromatographic method for the determination of aminoglycoside antibiotics was demonstrated.

Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard.

Abstract: To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 microL of mobile phase and 50 microL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 x 4.6 mm C(18) with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow-rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r(2) > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5-1000 ng/mL. The intra-run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were 80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS.

Analysis of methamphetamine and its metabolites in hair

Abstract: Methamphetamine (MA) is a sympathomimetic amine whose abuse has become a serious problem in Japan, Korea, Taiwan and other Southeast Asian countries. The use of hair for the determination of MA use has become more commonplace. The maximum period in which MA and its main metabolites (amphetamine and p-hydroxymethamphetamine) can be detected in urine is about 10 days after its use. However, proof of MA use is possible in hair even several years after its use if the part of the hair that grew in the period of its use is available. In addition, segmental analysis of hair is capable of clarifying the history of MA abuse. This paper reviews the clean-up, extraction, analytical method and distribution of MA and its metabolites in hair from reports published in the last 20 years.

Identification and determination of ecdysone and phenylpropanoid glucoside and flavonoids in Lamium maculatum by capillary zone electrophoresis

Abstract: The simultaneous determination of 20-hydroxy ecdysone (1), 3,7-dimethoxy-quercetin (2), acteoside (3) and rutin (4) in the mixture of leaf and stem, and the flower of Lamium maculatum has been investigated by capillary zone electrophoresis for the first time. With an electrolyte containing 30 mmol/L borate, at pH 9.47 and 20 kV applied voltage, the four active compounds were completely separated within 5 min with satisfactory results. The effects of concentration of borate and electrolyte pH on electrophoretic behavior and separation were studied. Regression equations revealed linear relationships (correlation coefficients 0.9998-0.9999) between the peak area of each analyte and the concentration. The levels of analytes in the different parts of Lamium maculatum were easily determined with recoveries ranging from 98.3 to 105.0%.

Molecular characterization of two host-guest associating hyaluronan derivatives.

Abstract: Molecular characteristics were determined of two high-molecular-weight water-soluble hyaluronan derivatives, namely β-cyclodextrin (HA-β-CD) and N-acylurea (EDC-HA). The weight-average molecular weight (Mw) of HA-β-CD and of EDC-HA, determined with a multi-angle light scattering detector connected on-line to a size exclusion chromatographic system, was respectively 185.3 and 86.8 kDa. However the Mw value determined for the equimolar mixture of the two HA derivatives equaled 556.0 kDa. Similarly, the gyration radius of the above equimolar mixture, Rg = 80.6 nm, was significantly greater than the values found for the single HA derivative, i.e. 40.2 nm for HA-β-CD and 23.8 nm for EDC-HA. These data indicate that the two kinds of substituents, bound to the polymeric chains, form host–guest/inclusion complexes resulting in polymacromolecular associates/aggregates. Copyright © 2003 John Wiley & Sons, Ltd.

An HPLC-MS method for simultaneous estimation of α, β-arteether and its metabolite dihydroartemisinin, in rat plasma for application to pharmacokinetic study

Abstract: This manuscript reports, the development and validation of a sensitive and selective assay method for simultaneous determination of alpha,beta-arteether and its metabolite dihydroartemisinin (DHA) in rat plasma by liquid chromatography-mass spectrometry. Chromatographic separations were achieved by gradient elution of the analytes with an initial composition of methanol-potassium acetate buffer (pH 4; 73:27, v/v) to 100% methanol in 3 min and maintained for 5 min on a Spheri-10, RP(18) (100 x 4.6 mm i.d.) column following an RP(18) (30 x 4.6 mm i.d.) guard column. The total effluent from the column was split so that one-tenth was injected into the electrospray LC/MS interface. ESI-MS analysis was performed using a Micromass Quattro II Triple Quadrupole Mass Spectrometer equipped with an electrospray source. The MS analysis was carried out at cone voltage of 22 V with a scan range of 200-500 Da. The analytes were quantified from the [M+ K](+) ion chromatograms of alpha,beta-arteether at m/z 352, DHA at m/z 323, artemisinin at m/z 321 and propyl ether analogue of arteether at m/z 365. Liquid-liquid extractions with a combination of n-hexane and hexane-ethyl acetate (8:2) were used to isolate alpha,beta-arteether and DHA from rat plasma. The method was validated and gave good accuracy and precision for the studied domain. Linearity in serum was observed over the range 4.375-70 ng/mL for alpha-arteether and 10-160 ng/mL for beta-arteether and DHA. Percentage bias (accuracy) and within- and between-assay precision were well within the acceptable range. This method was applied to study the pharmacokinetics following oral administration of alpha,beta-arteether (30 mg/kg) in rats.

Resolution of enantiomers of ketoprofen by HPLC: a review.

Abstract: Today, a heightened awareness of the applicability of enantiomers in medicine and clinical practice has been gene-rated due to the continuous evolvement of the field of chirality. In this context, this article provides a review of separation of ketoprofen, an important drug, in a popular class of non-steroidal anti-inflammatory drugs (i.e. profens). This review highlights various methodologies, logistical considerations for separation and provides an exhaustive list of applications mainly focusing on the pharmacokinetic aspects. Clearly, the application of enantioselective methods for drug racemates paves the way to understand the in vivo behavior of individual enantiomer and hence an opportunity for an alternate and/or better option for treating the disease.

Analysis of three effective components in Fructus corni and its preparations by micellar electrokinetic capillary chromatography

Abstract: An efficient micellar electrokinetic capillary chromatography method was developed to analyze three major active components including morroniside, loganin and gallic acid in Fructus corni and its six preparations for the first time. The factors that could affect the separation were studied, such as the pH of the buffer, concentrations of SDS, organic modifier and β-CD, and the applied voltage. The optimum analysis conditions were 10 mmol/L NaH2PO4–5 mmol/L Na2B4O7 (pH 6.8) buffer containing 140 mmol/L SDS, 1 mmol/L β-CD, 5% (v/v) methanol and 12.5 kV applied voltage. The linearity between the peak-areas and the concentrations of the analytes were investigated, and they exhibit excellent linear behavior over the concentration ranges (correlation coefficients 0.9953–0.9995). In addition, the pKa of gallic acid was determined using capillary zone electrophoresis. The result was consistent with that reported by the literature. Copyright © 2003 John Wiley & Sons, Ltd.

Direct injection HPLC method for the determination of selected phenothiazines in plasma using a Hisep column

Abstract: A direct plasma injection HPLC method has been developed for the determination of selected phenothiazines (promethazine, promazine, chlorpromazine) using a Hisep column. The method is easy to perform and requires 20 µL of a filtered plasma sample. The chromatographic run time is less than 11 min using a mobile phase of 15:85 v/v acetonitrile–0.18 m ammonium acetate pH 5.0 and UV detection at 254 nm. The method is linear in the concentration range 0.1–25 µg mL−1 (r > 0.99, n = 6) for each analyte with RSD less than 6%. Interday and intraday variability were found to be ≤14%. The limits of detection and quantitation were 0.1 (S/N > 3) and 0.25 µg mL−1 (S/N > 10), respectively, for each of the three phenothiazines. We can also apply this method to separate three other phenothiazines (ethopromazine, trifluoroperazine, prochlorperazine), although it lacks the selectivity to determine the concentration of all six drugs concurrently. The separation is feasible using these drugs in certain combinations. Copyright © 2003 John Wiley & Sons, Ltd.

Bioanalytical considerations for compounds containing free sulfhydryl groups.

Abstract: Thiol-containing compounds pose bioanalytical challenge in several dimensions due to extreme reactivity of the sulfhydryl group. The development of robust bioanalytical methodology for thiol groups should address the aspects of adequate stabilization in the biological matrix and selectivity considerations. In this context, availability of plethora of thiol reagents provides ample opportunity to achieve the above goals. However, several considerations need to be factored into the decision-making of a suitable scheme for the thiol drugs under investigation. This review provides some critical insights to many such considerations that may be vital for development and validation of methods for thiol-containing drugs.

Synthesis and analysis of aminochromes by HPLC-photodiode array. Adrenochrome evaluation in rat blood.

Abstract: The catecholamine oxidation process induces cardiotoxicity and neurotoxicity. Catecholamines can oxidize to aminochromes through autoxidation or by enzymatic or non-enzymatic catalysis. Although some toxic effects seem to be related to the formation of aminochromes there is still scarce information concerning the identification and evaluation of these compounds in in vivo models. In this study five catecholamines were oxidized to their respective aminochromes: adrenaline/adrenochrome; noradrenaline/noradrenochrome; dopa/dopachrome; dopamine/dopaminochrome; and isoproterenol/isoprenochrome. The evaluation of the catecholamines oxidation profile was performed by HPLC with photodiode array detection and using either enzymatic (tyrosinase) or non-enzymatic [Ag2O, CuSO4, NaIO4 and K3Fe(CN)6] catalytic systems. The NaIO4 was found to be the most efficient oxidant of catecholamines. An isocratic reverse-phase HPLC method was developed to analyse each pair of catecholamine–aminochrome. The analytical system was then applied to the detection of adrenochrome in rat blood at 490 nm. Thus, adrenochrome was administered i.p. to rats and its concentration in whole blood was monitored after 5, 15 and 25 min. Blood treatment for adrenochrome evaluation consists of an acidification for protein precipitation followed by a rapid neutralization. The results showed a rapid decrease of adrenochrome concentration in blood after its administration. The adrenochrome present in blood was characterized by UV and tandem mass spectrometry. Copyright © 2002 John Wiley & Sons, Ltd.

HPLC determination of diltiazem in human plasma and its application to pharmacokinetics in humans.

Abstract: A simple and sensitive reversed-phase high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of diltiazem in human plasma and the study of the pharmacokinetics of the drug in the human body. Diltiazem and diazepa (internal standard) were extracted with a mixed organic solution of hexane, chloroform and isopropanol (60:40:5, v/v/v), and then HPLC separation of the drugs was performed on an Spherisorb C18 column and detected by ultraviolet absorbance at 239 nm. The use of methanol–water solution (containing 2.8 mm triethylamine, 80:20, v/v) as the mobile phase at a flow-rate of 1.2 mL/min enables the baseline separation of the drugs free from interferences with isocratic elution. The method was linear in the clinical range 0–300 ng/mL and the lower limit of detection of diltiazem in plasma was 3 ng/mL. The range of percentage of relative standard deviation (%RSD) was from 3.5 to 6.8% for within-day analyses and from 6.2 to 8.4% for between-day analyses, respectively. The extraction recoveries of diltiazem from spiked human plasma (n = 5) at three concentrations were 91.4–104.0%. The method has been used to determine diltiazem in human plasma samples from eight volunteers who had taken diltiazem hydrochloride slow release tables and the data obtained was fitted with a program on computer to study the pharmacokinetics. The results showed that the peak level in plasma approximately averaged 118.5 ± 14.3 ng/mL at 3.1 ± 0.4 h, and the areas under the drug concentration curves (AUC) was 793.1 ± 83.1 ng.h/mL. Copyright © 2003 John Wiley & Sons, Ltd.

HPLC investigation of free and bound propofol in human plasma and cerebrospinal fluid.

Abstract: The paper compares the total propofol concentration in the cerebrospinal fluid (CSF) with the free drug concentration in plasma measured in 35 humans scheduled for elective neurosurgical procedures during propofol anaesthesia. The concentrations of total and free propofol in the blood and CSF samples were measured by means of HPLC using liquid-liquid extraction and ultrafiltration in the sample preparation procedure. The arterial blood and CSF samples (collected from intraventricular drainage) were taken at the same time. According to the obtained results, the usually expected equality between free drug concentration in plasma and its total concentration in CSF is not valid for propofol: the unbound propofol concentration in plasma is not equal to its total concentration in CSF (p < 0.05). This difference suggests a substantial contribution of active transport in propofol transfer from blood into CSF. Moreover, the paper shows the presence of bound propofol in CSF, which is a novel finding.

Determination of the compositions of polysaccharides from Chinese herbs by capillary zone electrophoresis with amperometric detection.

Abstract: In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was applied to determine the compositions of hetero-polysaccharides from Chinese herbs, Angelica sinensis and flax by analyzing their hydrolyzed monosaccharides: fucose, galactose, glucose, arabinose, rhamnose and xylose. Under the selected optimum conditions, the six monosaccharides could be perfectly separated within 25 min and showed significant current responses at copper electrodes. The linear ranges of the six monosaccharides were all from 5.0 x 10(-6) to 2.0 x 10(-4) mol L(-1) and their detection limits were lower or near 1.0 x 10(-6) mol L(-1) (S/N = 3). Experiments showed that the Angelica sinensis polysaccharide was composed of fucose, galactose, glucose, arabinose, rhamnose and xylose (mole ratio 1.0:13.6:15.0:8.7:21.3:3.7), and the flax polysaccharide was composed of galactose, glucose and arabinose (mole ratio 1.0:4.98:1.1). The purity of these polysaccharides leached by the introduced leaching method was 98.3 and 97.6%, respectively. Analyzing polysaccharides by this method has some merits of speed, simple instrumentation and operation, high sensitivity and high reproducibility.

Simultaneous quantification of budesonide and its two metabolites, 6β‐hydroxybudesonide and 16α‐hydroxyprednisolone, in human plasma by liquid chromatography negative electrospray ionization tandem mass spectrometry

Abstract: A sensitive, rapid and selective liquid chromatography negative electrospray ionization tandem mass spectrometry [LC-(−)ESI-MS-MS] method has been developed and validated for the simultaneous quantification of budesonide (BUD) and its major metabolites, 6β-hydroxybudesonide (OH-BUD) and 16α-hydroxyprednisolone (OH-PRED) in human plasma. The method was validated over a linear range from 0.1 to 10 ng/mL for all three analytes using a solid-phase extraction procedure with 9-fluoro-hydrocortisone as the internal standard. The between-day and within-day coefficients of variation for all compounds were ≤20% at the concentrations of lower limit of quantification and ≤15% at other quality control concentrations. The utility of this assay was demonstrated by monitoring BUD, OH-BUD and OH-PRED plasma concentrations in one healthy subject for 24 h following a 3 mg oral dose of budesonide, administered as a pH modified release capsule (Budenofalk®) to healthy volunteers. Copyright © 2003 John Wiley & Sons, Ltd.