Specific HPLC Assay Research Articles

Specific HPLC determination of toxic alkaloids in aconite roots compared with amount ot total alkaloids determined by titration

Aconite roots are known to be very toxic due to diterpene ester alkaloids [1]. Therefore, in Chinese medicine they are only used after processing. Hydrolysis of the ester groups decreases toxicity [2]. Nevertheless, several cases of poisoning by unprocessed or improperly processed aconite roots have been reported [3]. Recently we suggested a specific sample preparation method and HPLC assay for the analysis of toxic aconite alkaloids [4]. Now, an improved method with an even better resolution is presented. Using this method, we have determined the content of mesaconitine, aconitine and hypaconitine in 30 commercial samples of processed aconite roots. In most of the samples, toxic aconite alkaloids were not detectable, or only traces were found. However, in four samples we could detect more than 0.04% of hypaconitine and mesaconitine, the highest with a content of 0.16%. Therefore, this method is suggested as a purity test for the European Pharmacopoeia. Acute toxicity of batches high in hypaconitine and mesaconitine was also confirmed in CFLP mice. In the aconite monograph of the German Homeopathic Pharmacopoeia, alkaloids are determined by a titration method. We compared the results of HPLC analysis of toxic alkaloids (mesaconitine, aconitine and hypaconitine) with the results obtained by the titration method, and found no correlation. Samples which were lacking mesaconitine, aconitine and hypaconitine, still contained up to 0.2% alkaloids determined by titration. Therefore, titration of alkaloids is not appropriate as an assay for toxic alkaloids in aconite roots.

524 Oesophageal cancer proliferation is mediated by cytochrome P450 2C9 (CYP2C9)

A preliminary report implicated cytochrome P450 (CYP) 2C9 in the human liver microsomal O-demethylation of S-naproxen, suggesting that this pathway may be suitable for investigation of human hepatic CYP2C9 in vitro. Kinetic and inhibitor studies with human liver microsomes and confirmatory investigations with cDNA-expressed enzymes were undertaken here to define the role of CYP2C9 and other isoforms in the O-demethylation of R- and S-naproxen. All studies utilised a newly developed sensitive and specific HPLC assay that measured the respective O-desmethyl metabolites of R- and S-naproxen in incubations of human liver microsomes and in COS cell lysates. Microsomal R- and S-naproxen O-demethylation kinetics followed Michaelis-Menten kinetics, with respective mean apparent Km values of 123 μM and 143 μM. Sulfaphenazole, a specific inhibitor of CYP2C9, reduced the microsomal O-demethylation of R-and S-naproxen by 43% and 47%, respectively, and the CYP1A2 inhibitor furafylline decreased R- and S-naproxen O-demethylation by 38% and 28%, respectively. R,S-Mephenytoin was a weak inhibitor of R- and S-naproxen O-demethylation, but other CYP isoform specific inhibitors (e.g., coumarin, diethyldithiocarbamate, quinidine, troleandomycin) had little or no effect on these reactions. cDNA-expressed CYP2C9 and CYP1A2 were both shown to O-demethylate R- and S-naproxen. Apparent Km values (92–156 μM) for the reactions catalysed by the recombinant enzymes were similar to those observed for human liver microsomal R- and S-naproxen O-demethylation. The data demonstrate that CYP2C9 and CYP1A2 together account for the majority of human liver R- and S-naproxen O-demethylation, precluding the use of either R- or S-naproxen as a CYP isoform-specific substrate in vitro and in vivo.

Simultaneous determination of diosmin and diosmetin in human plasma by ion trap liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry: Application to a clinical pharmacokinetic study

Diosmetin (3′,5,7-trihydroxy-4′-methoxyflavone) is the aglycone of the flavonoid glycoside diosmin (3′,5,7-trihydroxy-4′-methoxyflavone-7-ramnoglucoside). Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the simultaneous determination of diosmin and diosmetin in human plasma was developed and validated. Plasma samples were incubated with β-glucuronidase/sulphatase. The analytes were isolated by liquid–liquid extraction with tert-butyl methyl ether at pH 2, and separated on a C 18 reversed-phase column using a mixture of methanol/1% formic acid (58:42, v/v) at a flow rate of 0.5 ml/min. APCI in the positive ion mode and multiple reaction monitoring (MRM) method was employed. The selected transitions for diosmin, diosmetin and the internal standard (7-ethoxycoumarin) at m/ z were: 609.0 → 463.0, 301.2 → 286.1 and 191, respectively. A good linearity was found in the range of 0.25–500 ng/ml ( R 2 > 0.992) for both compounds. The intra-batch assay precision (CV) for diosmin and diosmetin ranged from 1.5% to 11.2% and from 2.8% to 12.5%, respectively, and the inter-batch precision were from 5.2% to 11.5% and 8.5% to 9.8%, respectively. The accuracy was well within the acceptable range the accuracies (from −2.7% to 4.2% and −1.6% to 3.5% for diosmin and diosmetin, respectively). The mean recoveries of diosmin, diosmetin and the internal standard were 87.5%, 89.2% and 67.2%. Stability studies showed that diosmin and diosmetin were stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of diosmin in healthy volunteers following a single oral administration (Daflon ®).

Simultaneous determination of five systemic azoles in plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and automatable HPLC assay was developed for a simultaneous determination of systemic azoles (fluconazole, posaconazole, voriconazole, itraconazole and its metabolite hydroxyl-itraconazole, and ketoconazole) in plasma. The major advantage of this assay was sample preparation by a fully automatable solid phase extraction with Varian Plexa cartridges. C6-phenyl column was used for chromatographic separation, and UV detection was set at a wavelength of 260 nm. Linezolid was used as an internal standard. The assay was specific and linear over the concentration range of 0.05 to 40 μg/ml excepted for fluconazole which was between 0.05 and 100 μg/ml, and itraconazole between 0.1 and 40 μg/ml. Validation data for accuracy and precision for intra- and inter-day were good and satisfied FDA's guidance: CV between 0.24% and 11.66% and accuracy between 93.8% and 108.7% for all molecules. This assay was applied to therapeutic drug monitoring on patients hospitalized in intensive care and onco-hematologic units.

Bone turnover markers: a key tool for understanding osteoporosis

A search of the Web of Science database (http://apps. isiknowledge.com) for publications by Pierre Delmas indicates that he has 30 original publications concerning bone turnover markers out of the 67 publications with citation counts over 100. It is clear that his contribution to the field of bone turnover markers is great and bone turnover marker research has been a large fraction of his contribution to research into metabolic bone diseases. Dr. Delmas has contributed to many aspects of research into bone turnovermarkers. In a seminal paper, he was the first to use a specific HPLC assay to measure the 24-h excretion of pyridinoline and deoxypyridinoline in normal adults of both sexes, in patients with primary hyperparathyroidism, and in patients with Paget’s disease before and after intravenous pamidronate therapy. He concluded these assays were the first sensitive and specific bone resorption markers [1]. He sought to identify the sources of variation in these and other markers, such as the effects of age, menopause, gender, growth, exercise, season, and renal function, such that we could make better use of these markers. He validated these markers in studies of bone histomorphometry in osteoporosis and other metabolic bone diseases, as well as endocrine diseases and malignant bone disease. He examined their clinical utility in each of these disorders. He based his work not just on his very productive research INSERM unit in Lyon, but also on collaborations with scientists from around the world and with industry. The area to which he made the greatest contribution was to postmenopausal osteoporosis. He was able to demonstrate that high bone resorption that was a consequence of estrogen deficiency of menopause was associated with high rates of bone loss and increased risk of fracture; the contribution of these results to our ability to predict fracture is covered in the article in this issue by Dr. Melton. The detailed study of bone turnover markers in postmenopausal woman allowed Dr. Delmas to contribute to two major concepts that today we all take for granted. The first of these was that bone turnover markers remain elevated long after the menopause, a report from the OFELY study [1]. This was a population-based study of 693 women including 432 women who were 1 to 40 years past menopause. This was a comprehensive study of three bone formation markers (osteocalcin, bone alkaline phosphatase, and procollagen type I C-propeptide) and two bone resorption markers (Nand C-telopeptides of type I collagen). Dr. Delmas showed that bone turnover markers increased by 37% to 97% at the time of menopause and that the increase was associated with the onset of irregular menstruation and elevation in follicle-stimulating hormone. Bone formation markers remained elevated for up to 40 years after menopause. He adjusted the urinary excretion of bone resorption markers for total body bone mineral and reported that these too remained elevated for up to 40 years after menopause. He examined the relationship between bone turnover markers and bone mineral density and found only a weak association in premenopausal women, but a stronger relationship in postmenopausal women, especially those women who were 30 or more years after menopause, in whommarkers accounted for 52% of bone mineral density variance. This concept of high bone turnover and increased Osteoporos Int (2009) 20 (Suppl 3):S237–S238 DOI 10.1007/s00198-008-0695-y

Simultaneous determination of the major active components of tea polyphenols in rat plasma by a simple and specific HPLC assay

A simple and specific HPLC assay for simultaneous determination of two major active components (−) epigallocatechin-3-gallate (EGCG), and (−) epicatechin-3-gallate (ECG) of tea polyphenols (TP) in rat plasma was developed and validated. Following addition of resorcinol as internal standard (IS) the analytes were isolated from rat plasma by liquid–liquid extraction with ethyl acetate. The chromatographic separation was achieved on a reversed-phase C18 column using an isocratic mobile phase consisting of 0.1% citric acid + CH 3CN (86:14, v/v) running at flow rate of 1.5 mL/min. The effluent was monitored at a wavelength of 280 nm. EGCG, ECG and IS were well separated from each other and free from interference from blank plasma and other components in TP as well as metabolites post-dosing. The calibration curve was constructed by plotting peak area ratio of analytes to IS vs. concentration. The method showed good linearity over range of 0.5–300 μg/mL for EGCG and 0.1–60 μg/mL for ECG ( r > 0.999). The intra- and inter-day precision (R.S.D.) was better than 6 and 12%, respectively. Assay accuracy was better than 94.78% for both compounds. Extraction recovery at QC samples was between 85.73 and 91.93% for EGCG and 79.08 and 86.51% for ECG. The developed method was successfully used to simultaneously measure plasma concentrations of EGCG and ECG after intravenous administration of TP to rats and yielded two typical biexponential decay concentration–time curves.

Relationship between serum mono-hydroxy-carbazepine concentrations and adverse effects in patients with epilepsy on high-dose oxcarbazepine therapy

Purpose To investigate the relationship between the serum concentration of the mono-hydroxy-derivative (MHD) of oxcarbazepine (OXC) and adverse effects (AEs) in epileptic patients on high-dose OXC therapy. Patients and methods Forty-four consecutive patients, aged 18–65 years, with refractory epilepsy receiving OXC dosages ≥1500 mg/day (range 1500–3300 mg/day) were assessed at an outpatient clinic. Serum MHD concentrations were determined by a specific HPLC assay in samples collected before the morning dose and 2 h after drug intake. An independent observer assessed AEs at each sampling time. Results AEs were reported in five patients at the first sampling time, and in 26 patients at the second sampling time. Nystagmus, sedation, blurred vision, and dizziness were the most frequent AEs. MHD concentrations (means ± S.D.) associated with AEs were 29.6 ± 5.58 compared with 21.7 ± 5.0 mg/L when no AEs were detected ( p = 0.0001). AEs were minimized in most patients by reducing OXC dose, increasing the number of daily administrations, or both. Conclusion Patients with serum MHD concentrations ≥30 mg/L are at greater risk of developing AEs. In many patients, AEs occur intermittently in relation to fluctuations in serum MHD. Monitoring MHD concentrations could help in the management of patients on high-dose OXC therapy.

HPLC assay for bupropion and its major metabolites in human plasma

Bupropion is used clinically as an antidepressant and in smoking cessation. As it is metabolised to hydroxybupropion specifically by CYP2B6, bupropion has also been used as a probe to assess CYP2B6 activity. A specific and reproducible HPLC assay has been developed to simultaneously quantify bupropion and its major metabolites hydroxybupropion, threohydrobupropion and erythrohydrobupropion in human plasma. The analysis was performed on an Aqua C18 HPLC column, with a mobile phase consisting of 45:55 of methanol:0.05 M phosphate buffer (pH 5.5) and simultaneous UV detection at 214 nm (bupropion metabolites) and 254 nm (bupropion, internal standard timolol maleate). The assay showed a linear response for bupropion (2.5–250 ng/mL), threohydrobupropion (5–250 ng/mL), erythrohydrobupropion (10–250 ng/mL) and hydroxybupropion (10–1000 ng/mL). Extraction recovery was reproducible and greater than 55% for each analyte. The inter- and intra-day assay variability (measured as percent coefficient of variation; %CV) was less than 15% for all analytes. Limit of quantification was 2.5 ng/mL for bupropion, 5 ng/mL for threohydrobupropion and 10 ng/mL for hydroxybupropion and erythrohydrobupropion. This assay is more sensitive than currently published methods using HPLC with UV detection for the simultaneous quantitation of bupropion and metabolites and can be used for assessing CYP2B6 activity in vivo following a single dose of bupropion.

A validated HPLC determination of the flavone aglycone diosmetin in human plasma.

Diosmetin, 3',5,7-trihydroxy-4'-methoxy flavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti-inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7-ethoxycoumarin were isolated from plasma by liquid-liquid extraction and separated on a C8 reversed-phase column with methanol-water-acetic acid (55:43:2, v/v/v) as the mobile phase at 43 degrees C. Peaks were monitored at 344 nm. The method was linear in the 10-300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra-day and inter-day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%.

A validated solid-phase extraction HPLC method for the simultaneous determination of the citrus flavanone aglycones hesperetin and naringenin in urine

A simple, specific, precise, accurate, and robust HPLC assay for the simultaneous analysis of hesperetin and naringenin in human urine was developed and validated. Urine samples were incubated with β-glucuronidase/sulphatase and the analytes were isolated by solid-phase extraction using C18 cartridges and separated on a C8 reversed phase column using a mixture of methanol/water/acetic acid (40:58:2, v/v/v) at 45 °C. The method was found to be linear in the 50–1200 ng/ml concentration range for both hesperetin and naringenin ( r > 0.999). The accuracy of the method was greater than 94.8%, while the intra- and inter-day precision for hesperetin was better than 4.9 and 8.2%, respectively and for naringenin was better than 5.3 and 7.8%, respectively. Recovery for hesperetin, naringenin and internal standard 7-ethoxycoumarin was greater than 70.9%. The method has been applied for the determination of hesperetin and naringenin in urine samples obtained from a male volunteer following a single 300 mg oral dose of each of the corresponding flavanone glycosides hesperidin and naringin. The intra- and inter-day reproducibility through enzyme hydrolysis was less than 3.9% for both total (free + conjugated) hesperetin and naringenin. Stability studies showed urine quality control samples to be stable for both hesperetin and naringenin through three freeze–thaw cycles and at room temperature for 24 h ( error ≤ 3.6%).

Contamination of Aflatoxins in Herbal Medicinal Products in Thailand

Twenty-eight herbal medicinal products from Thailand were investigated for aflatoxin (AF) contaminations by employing a specific HPLC assay for the determination of AFB1, B2, G1 and G2. The samples were extracted with 80% (v/v) methanol in water before further cleaned up with an immunoaffinity column and followed by the detection of AFs by using an electrochemically post-column derivatization with iodine and fluorescence detector. The extraction procedure was optimized in order to obtain the best recovery. The method was successfully carried out with all the herbal products diversified as to compositions and dosage forms. The results revealed that five (18%) of herbal samples were contaminated with detectable amount of the total AFs ranging from 1.7 to 14.3 ng/g. The association between particular herbal/plant and the AF contaminated could not be determined due to the low frequency of positive samples. The contaminated products were those in tablet (4) and capsule (1) dosage forms. It was possible that the original fungal infection of these products may have been derived from either the crude herbal or other ingredients making these preparations, such as starch. In conclusion, none of the AF contaminated level found was above the current legislative level permissible in Thailand (20 ng/g). A word of caution, however, exporting some high AF-contaminated herbal products to countries where more stringent permissable level of aflatoxins exist could result in trade Barriers.

HPLC and LC–MS studies on stress degradation behaviour of tinidazole and development of a validated specific stability-indicating HPLC assay method

The objective of the current investigation was to study the degradation behaviour of tinidazole under different ICH recommended stress conditions by HPLC and LC–MS, and to establish a validated stability-indicating HPLC method. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal decomposition. Extensive degradation was found to occur in alkaline medium, under oxidative stress and in the photolytic conditions. Mild degradation was observed in acidic and neutral conditions. The drug was stable to thermal stress. Successful separation of drug from degradation products formed under stress conditions was achieved on a C-18 column using water–acetonitrile (88:12) as the mobile phase. The flow rate was 0.8 ml min −1 and the detection wavelength was 310 nm. The method was validated with respect to linearity, precision, accuracy, specificity and robustness. The utility of the procedure was verified by its application to marketed formulations that were subjected to accelerated stability studies. The method well separated the drug and degradation products even in actual samples. The products formed in marketed liquid infusions were similar to those formed during stress studies.

Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production.

beta-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce beta-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and beta-exotoxin production. A specific HPLC assay for type I beta-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of beta-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed beta-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as "common," "seldom," or "rare" beta-exotoxin producers. The serovar-dependent beta-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovar-dependent regulation of plasmid-encoded genes.

A validated high-performance liquid chromatographic method for the determination of glibenclamide in human plasma and its application to pharmacokinetic studies

Glibenclamide is a potent second generation oral sulfonylurea antidiabetic agent widely used for the treatment of type II diabetes melitus. A rapid, sensitive, precise, accurate and specific HPLC assay for the determination of glibenclamide in human plasma was developed and validated. After addition of flufenamic acid as internal standard, the analytes were isolated from human plasma by liquid-liquid extraction. The method was linear in the 10-400 ng/ml concentration range (r > 0.999). Recovery for glibenclamide was greater than 91.5% and for internal standard was 93.5%. Within-day and between-day precision, expressed as the relative standard deviation (RSD%), ranged from 1.4 to 5.9% and 5.8 to 6.6%, respectively. Assay accuracy was better than 93.4%. The assay was used to estimate the pharmacokinetics of glibenclamide after oral administration of a 5 mg tablet of glibenclamide to 18 healthy volunteers.

Limited Phase I Study of Morphine-3-Glucuronide

The toxicity of morphine-3-glucuronide (M3G) has been investigated in an open, uncontrolled, single-blinded, single dose study over a limited range of doses. Three cohorts each of three healthy volunteers received 7.5, 15, and 30 mg/70 kg intravenous (IV) M3G. Blood sampling was undertaken for the following 24 h. Subjective toxicity was recorded on visual analogue scales and plasma M3G concentrations measured by a specific HPLC assay. Virtually no effects and no change in cardiovascular or respiratory parameters were seen. The pharmacokinetics fitted a two-compartment model. The mean elimination half-life (+/- S.D.) of M3G was 1.66 (+/- 0.47) h. Mean AUC standardized to a dose of 1 mg/70 kg was 228 (+/- 62) etamolL(-1) x h. Mean M3G clearance was 169 (+/- 48) mLmin(-1) and the mean volume of distribution was 23.1 (+/- 4.8) liters. At the doses investigated there were no clear neuroexcitatory effects, no opioid effects, and the pharmacokinetics were very similar to those of morphine-6-glucuronide (M6G).

Selective radioprotection of hepatocytes by systemic and portal vein infusions of amifostine in a rat liver tumor model

Purpose: The tolerance of the liver to radiation is too low to permit an effective dose to be delivered to patients who have diffuse intrahepatic cancer. In this study we evaluated whether systemic or portal venous administration of the aminothiol compound, amifostine, could protect the normal liver from the effects of ionizing radiation without compromising tumor cell kill in a rat liver tumor model. Methods and Materials: Rats implanted with liver tumors were infused with 200 mg/kg amifostine over 15 min via the femoral or portal vein. The livers were irradiated with a single 6-Gy fraction 15–20 min after the termination of amifostine infusion. Protection of the liver was assessed by an in vitro hepatocyte micronucleus assay and tumor protection by an in vivo- in vitro clonogenic survival assay. Tissue levels of the active metabolite, free WR-1065, were determined in the tumor and in the normal liver using a specific HPLC assay with electrochemical detection. Results: After a 6-Gy fraction, the frequency of hepatocyte micronuclei after administration of saline, systemic amifostine, and portal venous amifostine was 18.7 ± 1%, 6.8 ± 1%, and 9.9 ± 2%, respectively, corresponding to a radiation equivalent effect of 6 Gy ± 0.5, 1.8 Gy ± 0.3, and 2.5 Gy ± 1.3, respectively. Both amifostine conditions showed considerably less radiation effect than saline-treated controls ( p < 0.01); the two amifostine conditions did not differ ( p = 0.3). The surviving fraction of tumor cells was not affected by amifostine treatment and was 0.03 ± 0.02 and 0.05 ± 0.03 for systemic and portal venous delivery and 0.06 ± 0.02 for control animals (ANOVA analysis showed no significant difference of the means p = 0.34). Portal venous delivery produced significantly less WR-1065 in the tumor compared to systemic administration (54 μM ± 36 vs. 343 μM ± 88, respectively, p = 0.03). Conclusions: Both systemic and portal venous administration of amifostine effectively protect hepatocytes from ionizing radiation without compromising tumor cell kill in a clinically relevant animal model. These findings suggest that amifostine may be a selective normal tissue radioprotectant in liver cancer and that regional/portal infusions may be preferable to systemic dosing.

Thermal inactivation kinetics of bovine cathepsin D.

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6.7) and skimmed milk of 6.5 and 7.6 degrees C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 degrees C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.

An HPLC assay and a microbiological assay to determine levofloxacin in soft tissue, bone, bile and serum.

A simple, specific and sensitive HPLC assay for levofloxacin in serum, bile, soft tissue and bone was evaluated and validated. The samples were prepared by protein precipitation with acids and methanol, which yielded high recoveries (for serum and bile>98% and for bone and soft tissue>90%). The compounds were separated on a reversed phase column with an acidic mobile phase containing triethylamine. The eluate was monitored by fluorescence detection. The HPLC assay is linear over the usable concentration range (0.1-40 microg/ml) and it provides good validation data for accuracy and precision. Although comparison of HPLC results to the results of a microbiological assay showed congruent results (regression coefficients>0.967). HPLC should be the method of choice for determination of levofloxacin in biological matrices.

High-performance liquid chromatographic analysis of the tyrphostin AG1478, a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in mouse plasma

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is undergoing evaluation as a potential new anticancer agent. We have developed a specific and sensitive reversed-phase HPLC assay for AG1478 in mouse plasma. The method involves a rapid and simple extraction process followed by separation on a Symmetry C 8 stationary phase with a gradient of acetonitrile in ammonium acetate buffer. A linear response was achieved over the concentration range of 0.2–100 μ M using multilevel calibration with internal standard method of calculation. Inter- and intra-assay accuracy and precision were better than ±10%. The limit of quantitation was 0.2 μ M. We have used this method to study the preclinical pharmacokinetics of this new agent in mice.

High-performance liquid chromatographic assays for a second-generation novel oral iron chelator (APCP363) and their application to pharmacokinetic studies in rats

Sensitive and specific HPLC assays for APCP363 in biological matrices (rat plasma, urine and feces) were developed. The recovery of APCP363 ranged from 81.2 to 99.9% in plasma, from 82.1 to 92.8% in urine, and from 65 to 68% in feces. Standard deviations were below 10% for all analyses. The limits of quantitation were 0.1, 10 and 30 μg/ml in plasma, urine and feces, respectively. The HPLC assays, which are the first reports for APCP363 analysis in biological matrices, have been successfully applied to preliminary pharmacokinetic studies in rats. The stool assay is the first non-radiolabeled method for hydroxypyridinones in feces.