Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

Determination of nortriptyline in human serum by fully automated solid-phase extraction and on-line high-performance liquid chromatography in the presence of antipsychotic drugs

A fully automated on-line method for determination of nortriptyline in human serum was developed using an ASPEC XL (Gilson) solid-phase extraction apparatus in combination with high-performance liquid chromatography. Solid phase extraction was performed on cyanopropyl cartridges. HPLC was carried out using a C₁₈ column with a mobile phase of acetonitrile–0.01 M triethylamine (34:66 v/v) buffer, pH 3.0. UV detection was at 242 nm. The Inter-day CV% was <5%. Comparison with liquid–liquid extraction of serum from patients treated with nortriptyline showed good agreement. Studies of analytical interference from coadministered psychoactive drugs revealed that only imipramine and a methotrimeprazine metabolite interfered.     

Determination of centpropazine and its metabolite in rat serum by high-performance liquid chromatography and fluorescence detection

A new HPLC assay method was developed for the simultaneous assay for centpropazine (antidepressant) and its hydroxylated metabolite (II) to assess their pharmacokinetics and metabolism characteristics. Rat serum samples were extracted with ether, backwashed with n-hexane and injected onto the HPLC system, which used a C₁₈ column, gradient elution and fluorescence detection at 250 Ex/350 nm Em. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of <±20% at low and <±15% at higher concentrations. Samples were stable in autosampler prior to injection and after multiple freeze–thaw cycles. Linearity was observed between 0.625 and 20 ng/ml for both I and II in serum. Overall the method developed was highly sensitive and could be employed for a wide range of studies.     

Rapid quantitation of (−)-2′-deoxy-3′-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (−)-2′-deoxy-3′-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C₁₈ column using a mixture of phosphate buffer and methanol (88.3:11.7, v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 μl of serum. The standard curve was linear within the range of 20–10 000 ng/ml. Replicate analysis of three quality control samples (40–1500 ng/ml) led to satisfactory intra- and itner-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from −6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55°C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2–5 fmol/injection (S/N=3). Pro–Hyp, Pro–Gly and Pro–Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5–7.9 and 2.4–10.8%, respectively. The recoveries were in the range of 90.8–97.3%. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human serum (n=10) were 0.64±0.35, 0.078±0.047, 0.022±0.016, 177.0±43.0 and 11.1±3.5 μM, respectively. The concentrations of Pro–Hyp and Pro–Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.     

Determination of plasma phenobarbital concentration by high-performance liquid chromatography in rat offspring

Plasma phenobarbital (PB) concentrations in rat offspring were determined using a 9 μl capillary by high-performance liquid chromatography (HPLC). Capillary plasma which was put into a Bond Elut^® cartridge column by using 1 ml of 0.01 M KH₂PO₄ was applied to the column with 50 μl of 2 μg/ml of acetanilide (internal standard, I.S.). After washing the column, PB and I.S. were eluted with methanol and injected into the HPLC system. There were excellent linear correlation between the amount of PB and length of the capillary at three different concentrations. Calibration for PB was linear in the range of 0–50 μg/ml. The coefficients of variation were 3.4–5.0% and 5.9–7.5% in the within-day and between-day assays, respectively. The extraction recovery rates were 87.5–105.4%. By this method, it was possible to measure plasma PB concentrations in rat offspring without killing. These results suggested that this method is very useful to determine the plasma PB concentration derived from mother’s milk in newborn rats.     

Determination of olanzapine in human breast milk by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic–electrochemical assay was developed and validated for the quantification of olanzapine in human breast milk. The assay involved a solid-phase extraction (SPE) of olanzapine and its internal standard on a Bond Elut Certify LRC mixed-mode cartridge. After conditioning of the SPE cartridge, human milk (1 ml) was passed through the cartridge. The cartridge was washed with five separate washing steps to remove endogenous compounds, and the analytes were eluted with ethyl acetate–ammonium hydroxide (98:2, v/v) solution. The eluate was evaporated to dryness (gentle stream of nitrogen at 40°C), and the residue was dissolved in mobile phase. The extract was injected onto a YMC basic column (150 mm×4.6 mm I.D., 5 μm particle size) at a flow-rate of 1 ml/min. A mixture of 75 mM phosphate buffer, pH 7.0–acetonitrile–methanol (48:26:26, v/v/v) was used as the mobile phase. Standard curves with a lower limit of quantitation of 0.25 ng/ml of olanzapine were linear (r²≥0.9992) over a range of 0.25–100 ng/ml. Based on the analysis of quality control (QC) samples, the average inter-day accuracy (RE) was 99.0% with an average precision (CV) of 6.64% over the entire range. The stability of olanzapine in human milk was established after three freeze–thaw–heat cycles and storage at −70°C for 10 months. The validated method was used to measure olanzapine concentrations in human milk during a clinical trial.     

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated β-cyclodextrin as chiral mobile phase additive and solid-phase extraction

A sensitive and stereospecific HPLC method was developed for the analysis of (−)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-β-cyclodextrin (S-β-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)–absolute ethanol (80:20, v/v) containing 10 mM S-β-CD at a flow-rate of 0.7 ml/min. Recoveries of (−)- and (+)-pentazocine were in the range of 91–93%. Linear calibration curves were obtained in the 20–400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9–7.0% and 1.2–6.2%, respectively, for the (−)-enantiomer and 0.8– 7.6% and 1.2–4.6%, respectively, for the (+)-enantiomer (n=3).     

Determination of lamotrigine in human serum by liquid chromatography

A rapid high-performance liquid chromatographic method was developed using a short silica column (30 mm×4.6 mm) with an aqueous methanol mobile phase consisting of methanol–water–NH₄H₂PO₄ (94:5.96:0.04) adjusted to a final apparent pH of 5.0 and pumped at a flow-rate of 1 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm, and serum samples were prepared for HPLC analysis by extraction into dichloromethane after basification. Lamotrigine was eluted at 0.96 min. Within-day variation of the method was 4.46% at 0.75 μg/ml and 2.37% at 6.0 μg/ml, and day-to-day variation was 9.10% at 0.75 μg/ml and 7.28% at 6.0 μg/ml.     

High-performance liquid chromatographic assay for minocycline in human plasma and parotid saliva

A sensitive and specific high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of minocycline in human plasma and parotid saliva samples. Samples were extracted using an Oasis™ HLB cartridge and were injected into a C₈ Nucleosil column. The HPLC eluent contained acetonitrile–methanol–distilled water–0.1% trifluoroacetic acid (25:2:72.9:0.1, v/v). Demeclocycline was used as internal standard. The assay showed linearity in the tested range of 0.1–25 μg/ml. The limit of quantitation was 100 ng/ml. Recovery from plasma or parotid saliva averaged 95%. Precision expressed as %CV was in the range 0.2–17% (limit of quantitation). Accuracy ranged from 93 to 111%. In the two matrices studied at 20 and 4°C, rapid degradation of the drug occurred. Frozen at −30°C, this drug was stable for at least 2 months, the percent recovery averaged 90%. The method’s ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.     

Simultaneous determination of seven penicillins in muscle, liver and kidney tissues from cattle and pigs by a multiresidue high-performance liquid chromatographic method

A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction (SPE) was developed for determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in muscle, liver and kidney tissues of pigs and cattle. The compounds were extracted in aqueous solution by precipitation of organic materials with a mixture of sulphuric acid and sodium tungstate. The extract was cleaned up by SPE on a divinylbenzene-co-N-vinylpyrrolidone polymeric sorbent. Further clean-up was performed by liquid–liquid partition with diethyl ether. The extract was derivatised with benzoic anhydride and 1,2,4-triazole mercury (II) reagent. Chromatography was performed by reversed-phase gradient HPLC on a C₁₈ column with ultraviolet detection at 323 nm. The limits of detection estimated by a conservative model were in the range 8.9–11.1 μg/kg for amoxicillin, penicillin G, ampicillin, oxacillin, cloxacillin and nafcillin and 18.3–20.9 μg/kg for dicloxacillin. The mean recovery range was 66–77% for amoxicillin, 73–75% for penicillin G, 81–82% for ampicillin, 73–76% for oxacillin, 74–75% for cloxacillin, 66–72% for nafcillin and 58–65% for dicloxacillin.     

Biochemical properties of pigeon milk and its effect on growth

Summary The avian juvenile food pigeon milk was studied for its chemical composition and effect on growth in vivo and in vitro. Pigeon milk on a wet weight basis consisted of 9–13% protein, 9–11% fat, 0.9–1.5% carbohydrate, 0.8–1.1% ash, 0.10–0.12% non-protein nitrogen, energy content 5.6–6.8 kcal·g^-1. Except for proteins there was little or no decrease in pigeon milk constitutents during the first week of secretion. Pigeon milk proteins consisted of trichloroacetic acid (precipitable), trichloroacetic acid (soluble), and free amino acid components in the ranges 8.4–12.1%, 0.5–0.7% and 1.4–2.5%, respectively; whereas the level of trichloroacetic acid (precipitable) and trichloroacetic acid (soluble) components decreased by about 30%, that of the free amino acids increased by 9% in the first week. About 0.6–1.0% of pigeon milk sugar was found in the trichloroacetic acid (soluble) fraction and increased by 67% in the first week. The remainder was found in the trichloroacetic acid (precipitable) fraction and did not change during this period. Major lipids of pigeon milk were the neutral lipids (7.8–8.4%); the minor lipids were glycolipids (0.9–1.6%), phospholipids (0.5–1.4%) and cholesterol (0.5–0.6%). Squabs fed pigeon milk increased their body weight by 22-fold in the first 3 weeks after hatching, and crude extracts of pigeon milk stimulated the growth of cultured hamster ovary cells. These results reflect the ability of pigeon milk to stimulate growth both in vivo and in vitro.Abbreviations AOAC association of official analytical chemists - BRIT board of radiation and isotope technology - CHO chinese hamster ovary - DNA deoxyribonucleic acid - EDTA ethylenediaminetetra-acetic acid - FCS foetal calf serum - GF growth factor - GS goat serum - MEM minimum essential medium - NPN nonprotein nitrogen - PBS phosphate-buffered saline - PM pigeon milk - TCA(P) trichloroacetic acid precipitable fraction - TCA(S) trichloroacetic acid soluble fraction     

Determination of plasma concentrations of epirubicin and its metabolites by high-performance liquid chromatography during a 96-h infusion in cancer chemotherapy

In order to determine epirubicin and its metabolites at low concentrations (<38 ng/ml) in small plasma samples, a fast reliable method based on a precipitation pre-treatment and sensitive reversed-phase isocratic HPLC has been developed and validated for epirubicin in the range 5–100 ng/ml. The R.S.D. was 5–9% over this concentration range. For human serum containing 25 ng/ml of epirubicin, the inter- and intra-day variation was <10%. Recoveries of the metabolites epirubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone at 20 ng/ml ranged from 94–104%. The assay has been used to study human plasma samples taken during a 96-h infusion of epirubicin in a patient with multiple myeloma. The combined levels of the unseparated metabolites, epirubicin glucuronide and epirubicinol glucuronide, were semiquantitatively determined after treatment with β-glucuronidase. The metabolites epirubicinol and 7-deoxydoxorubicinolone, but not 7-deoxydoxorubicinone, were also detected and measured.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Enantioselective high-performance liquid chromatographic determination of nicardipine in human plasma

A sensitive method for the enantioselective high-performance liquid chromatography (HPLC) determination of nicardipine in human plasma is described. (+)-Nicardipine, (−)-nicardipine and (+)-barnidipine as an internal standard are detected by an ultraviolet detector at 254 nm. Racemic nicardipine in human plasma was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction. The extraction samples were purified and concentrated on a pre-column using a C₁ stationary phase and the enantiomers of nicardipine are quantitatively separated by HPLC on a Sumichiral OA-4500 column, containing a chemically modified Pirkle-type stationary phase. Determination of (+)- and (−)-nicardipine was possible in a concentration range of 5–100 ng ml⁻¹ and the limit of detection in plasma was 2.5 ng ml⁻¹. The recoveries of (+)- and (−)-nicardipine added to plasma were 91.4–98.4% and 93.3–96.7%, respectively, with coefficients of variation of less than 9.0 and 9.4% respectively. The method was applied to low level monitoring of (+)- and (−)-nicardipine in plasma from healthy volunteers.     

Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.     

Multiresidue analysis of β2-agonists in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection

Various β₂-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four β-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The β-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the β-agonists were eluted with ammoniated ethanol–hexane. The extract was analysed with an HPLC method with electrochemical detection. The β-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90–105% vs. 65–75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that β₂-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.