Semi-automatic liquid chromatographic analysis of pamidronate in serum and citrate plasma after derivatization with 1-naphthylisothiocyanate

The semi-automatic method for the determination of the bisphosphonate pamidronate in serum and citrate plasma involves a manual protein precipitation with trichloroacetic acid and a manual coprecipitation of the bisphosphonate with calcium phosphate, followed by an automated solid-phase extraction on anion-exchange columns. After off-line evaporation of the extract under nitrogen and reconstitution in water, the automatic procedure is continued by automatic derivatization with 1-naphthylisothiocyanate, ion-pair liquid–liquid extraction and a treatment with hydrogen peroxide, prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The intra- and inter-day precisions are 1.3 and 7%, respectively, for a standard of 100 ng ml⁻¹ pamidronate in serum; the average accuracy for this standard is 107%. The lower limit of quantification is 20 ng ml⁻¹ pamidronate in 1 ml of human serum.     

Intravenous Pamidronate is Associated with Reduced Mortality in Patients with Chronic Critical Illness

Objective: Chronic critical illness (CCI), characterized by prolonged mechanical ventilation and tracheostomy, commonly manifests with elevated bone resorption, which has previously been shown to abate after treatment with intravenous (IV) bisphosphonates. Our study assessed the impact of pamidronate administration on clinical outcomes in a CCI cohort.Methods: A retrospective case series was performed on 148 patients admitted to The Mount Sinai Hospital Respiratory Care Unit (RCU) from 2009–2010. We identified patients with CCI who did (n = 30) or did not (n = 118) receive IV pamidronate (30 to 90 mg). Both groups included patients with normal and abnormal renal function. Pamidronate was administered for elevated urine or serum N-telopeptide, hypercalciuria, or hypercalcemia.Results: RCU and 1-year mortality were significantly lower in the pamidronate group (0 and 20%, respectively) compared to nonreceivers (19 and 56%, respectively) (P = .0077 and P = .0004, respectively). After adjusting for differences in baseline creatinine, estimated glomerular filtration rate, and serum calcium, the association with reduced mortality remained significant at 1 year (P = .0132) and with borderline significance for RCU mortality (P = .0911). Creatinine was significantly lower 7 days following pamidronate administration (P = .0025), with no significant difference at 14 days compared to baseline. Pamidronate receivers showed a greater increase in albumin during the RCU stay (2.49 to 3.23 g/dL), compared to nonreceivers (2.43 to 2.64 g/dL) (P = .0007). Pamidronate administration was associated with a significantly reduced rate of hypoglycemia compared to RCU patients not receiving pamidronate (0.09 versus 0.12; P = .0071).Conclusion: Pamidronate use in a CCI population is associated with reduced mortality, lower hypoglycemia rates, improved albumin, and stable renal function.Abbreviations:BMI = body mass indexCCI = chronic critical illnessCI = confidence intervalCKD = chronic kidney diseaseCTx = C-telopeptideeGFR = estimated glomerular filtration rateICU = intensive care unitIV = intravenousNTx = N-telopeptidePMV = prolonged mechanical ventilationRCU = respiratory care unit     

Metabolic effects of single-dose pamidronate administration in prostate cancer patients with bone metastases

BACKGROUND: Increased osteolysis usually accompanies sclerotic bone metastases from prostate cancer. This provides a rationale for the use of bisphosphonates to treat bone pain and prevent skeletal complications. METHODS: The fasting urinary levels of calcium, hydroxyproline (OHPRO), pyridinolines (PYD), deoxypyridinolines (DPYD), collagen cross-linked N-telopeptide (NTX) and the serum values of calcium, total alkaline phosphatase and relevant bone isoenzyme, bone gla protein (BGP), carboxy-telopeptide of type I collagen (ICTP) and parathyroid hormone (PTH) were determined at baseline and on the 15th, 30th, 60th and 90th days after single-dose (90 mg) pamidronate administration in 35 consecutive prostate cancer patients with bone metastases. These biochemical indices and serum interleukin 6 (IL-6) were also measured after four days in the last consecutive 17 cases. RESULTS: PYD, DPYD and NTX showed a significant decrease lasting four weeks (p<0.01, <0.01 and <0.001, respectively). OHPRO and ICTP did not change significantly. The NTX decline was greater than that of PYD and DPYD (maximum percent decrease: -71.3, -23.1 and -28.2, respectively). Bone formation markers and serum calcium did not change significantly. Serum PTH showed a rapid initial increase followed by a slow decrease (p<0.001). DPYD and NTX patterns did not correlate with changes in bone pain. As observed in the last 17 cases, the maximum osteolysis inhibition after pamidronate occurred on the fourth day after drug infusion. Serum IL-6 levels showed a short-lived decrease preceded by a transient rise on the fourth day. CONCLUSIONS: Pamidronate is able to induce a decrease in bone resorption without significantly influencing bone formation. The maximum decrease in bone resorption occurs very early. NTX is the most sensitive bone resorption marker in bisphosphonate therapy monitoring. Changes in IL-6 but not bone resorption markers may be useful in the prediction of symptomatic response.     

Pamidronate: Treatment for severe hypercalcemia in neonatal subcutaneous fat necrosis

BACKGROUND: Subcutaneous fat necrosis (SCFN) of the newborn is an uncommon disorder that occurs in the first weeks of life after foetal distress. It can be complicated by potentially life-threatening hypercalcemia. Treatments of hypercalcemia have included hydration, furosemide and corticosteroids. Only one report has described the use of intravenous bisphosphonates for this condition. We propose that pamidronate could be the first line therapy for severe hypercalcemia in SCFN. PATIENTS AND RESULTS: Four newborns presented between 2001 and 2004 with SCFN complicated by severe hypercalcemia. At diagnosis, ionized calcium levels were higher than 1.4 mmol/l and were associated with high urinary calcium/creatinine ratios and high 1,25-dihydroxyvitamin D levels. Despite treatment with IV fluids, low calcium diet and furosemide, calcium levels remained high. The patients were given 3-4 doses (0.25-0.50 mg/kg/dose) of pamidronate. Urinary calcium/creatinine ratios and calcium levels decreased within 48-96 h. 1,25-dihydroxyvitamin D levels normalized with resolution of the skin lesions. No persistent nephrocalcinosis was observed. CONCLUSION: Pamidronate is effective, well-tolerated in the short-term and obviates the need for prolonged treatment with furosemide and corticosteroids. To prevent nephrocalcinosis, pamidronate might be considered as first line treatment for severe hypercalcemia in SCFN.     

Simultaneous determination of cytosine arabinoside,daunorubicin and etoposide in human plasma

A method for simultaneous bioanalysis of the three cytotoxic drugs cytosine arabinoside, daunorubicin and etoposide in human plasma was developed and validated. A HPLC method with ultra-violet and fluorescence detection, preceded by mixed-mode cation-exchange solid phase extraction sample preparation, was used for the quantification of the analytes. The assay was used for the simultaneous measurement of cytosine arabinoside, daunorubicin and etoposide with linearity in the ranges of 13–1500 ng/mL, 15–1000 ng/mL and 52.5–3500 ng/mL, respectively. The chromatographic run-time was 15.5 min. The overall precision (% relative standard deviation) was within 0.2–13.5% and the recovery ranged between 86.1% and 110.1% for the three drugs at all concentrations tested. Plasma samples were stable for at least two months when stored at −20 °C. The method was successfully applied to quantification of the three drugs in blood samples from patients undergoing induction treatment for acute myeloid leukaemia, thus demonstrating its suitability for clinical studies.     

Pamidronate in the treatment of bone metastases from breast cancer

Bone metastases afflict over 70% of patients with advanced breast cancer, resulting in impaired quality of life and significant clinical problems. Until appearance of the bisphosphonates there was no specific therapeutic treatment available to manage the symptoms of osteolytic bone metastases. Bisphosphonates are stable chemical analogues of pyrophosphate, and inhibit osteoclast-mediated bone resorption, the treatment is effective in reducing skeletal morbidity in breast cancer with fewer skeletal related events, reduced pain and analgesic consumption, and improved quality of life. As a result, bisphosphonates should now be part of the routine management of metastatic bone disease and multiple myeloma. Promising data have resulted in considerable interest in the possible adjuvant use of bisphosphonates. Pamidronate is an easy to use potent inhibitor of osteolysis, given in conjunction with standard anticancer therapies effectively relieves bone pain and improves performance status. Monthly pamidronate infusions for one or two years in addition to standard anticancer therapy reduce by more than one third the yearly frequency of skeletal-related events. The authors report their practice in which 119 breast cancer patients metastatic to bone received 90-120 mg pamidronate infusion/cycle in addition to standard breast cancer therapy every 3-4 weeks.     

Alendronate and Pamidronate calcium phosphate bone cements: setting properties and in vitro response of osteoblast and osteoclast cells

We have investigated the effect of Alendronate and Pamidronate, two bisphosphonates widely employed for the treatment of pathologies related to bone loss, on the setting properties and in vitro bioactivity of a calcium phosphate bone cement. The cement composition includes α-tricalcium phosphate (α-TCP) (90 wt%), nanocrystalline hydroxyapatite (5 wt%) and CaHPO₄ · 2H₂O (5 wt%). Disodium Alendronate and disodium Pamidronate were added to the liquid phase (bidistilled water) at two different concentrations: 0.4 and 1 mM (AL0.4, AL1.0, PAM0.4, PAM1.0). Both the initial and the final setting times of the bisphosphonate-containing cements increase with respect to the control cement. X-ray diffraction analysis, mechanical tests, and SEM investigations were carried out on the cements after different times of soaking in physiological solution. The rate of transformation of α-TCP into calcium deficient hydroxyapatite, as well as the microstructure of the cements, is not affected by the presence of Alendronate and Pamidronate. At variance, the bisphosphonates provoke a modest worsening of the mechanical properties. MG63 osteoblasts grown on the cements show a normal morphology and biological tests demonstrate very good rate of proliferation and viability in every experimental time. In particular, both Alendronate and Pamidronate promote osteoblast proliferation and differentiation, whereas they inhibit osteoclastogenesis and osteoclast function.     

Calcium Carbonate Toxicity: The Updated Milk-Alkali Syndrome; Report of 3 Cases and Review of the Literature

ObjectiveTo describe 3 patients with calcium carbonate-induced hypercalcemia and gain insights into the cause and management of the milk-alkali syndrome.MethodsWe report the clinical and laboratory data in 3 patients who presented with severe hypercalcemia (corrected serum calcium ≥ 14 mg/dL) and review the pertinent literature on milk-alkali syndrome.ResultsThe 3 patients had acute renal insufficiency, relative metabolic alkalosis, and low parathyroid hormone (PTH), PTH-related peptide, and 1,25-dihydroxyvitamin D concentrations. No malignant lesion was found. Treatment included aggressive hydration and varied amounts of furosemide. The 2 patients with the higher serum calcium concentrations received pamidronate intravenously (60 and 30 mg, respectively), which caused severe hypocalcemia. Of the 3 patients, 2 were ingesting acceptable doses of elemental calcium (1 g and 2 g daily, respectively) in the form of calcium carbonate. In addition to our highlighted cases, we review the history, classification, pathophysiologic features, and treatment of milk-alkali syndrome and summarize the cases reported from early 1995 to November 2003.ConclusionMilk-alkali syndrome may be a common cause of unexplained hypercalcemia and can be precipitated by small amounts of orally ingested calcium carbonate in susceptible persons. Treatment with hydration, furosemide, and discontination of the calcium and vitamin D source is adequate. Pamidronate treatment is associated with considerable risk for hypocalcemia, even in cases of initially severe hypercalcemia. (Endocr Pract. 2005;11: 272-280)     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

Clinical Efficacy and Safety of Pamidronate Therapy on Bone Mass Density in Early Post-Renal Transplant Period: A Meta-Analysis of Randomized Controlled Trials

Introduction

The overall effect of pamidronate on bone mass density (BMD) in the early renal transplant period varies considerably among studies. The effects of pamidronate on graft function have not been determined.

Materials and Methods

A comprehensive search was conducted in PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL) and Embase independently by two authors. Randomized controlled trials of pamidronate evaluating bone loss in the first year of renal transplantation were included. Methods reported in the “Cochrane Handbook for Systematic Reviews of Interventions 5.0.2” were used to evaluate changes of lumbar spine and femoral neck BMD, and serum creatinine, calcium and intact parathyroid hormone (iPTH) levels. Fixed or random effect models were used as appropriate.

Results

Six randomized trials evaluating 281 patients were identified. One hundred forty-four were treated with pamidronate and 137 were control patients. Administration of pamidronate was associated with significant reduction of bone loss in the lumbar spine, compared to the control group (standardized mean difference (SMD)  = 24.62 [16.25, 32.99]). There was no difference between the pamidronate treated and control femoral neck BMD (SMD  = 3.53 [−1.84, 8.90]). A significant increase in the serum creatinine level of the intervention group was seen, compared to the control group. The serum calcium and iPTH of the pamidronate and control groups were not different after 1 year (serum creatinine: SMD  = −3.101 [−5.33, −0.89]; serum calcium: SMD  = 2.18 [−0.8, 5.16]; serum iPTH: SMD  = 0.06 [−0.19, 0.31]). Heterogeneity was low for serum calcium and iPTH and high for serum creatinine.

Conclusions

This meta-analysis demonstrated the beneficial clinical efficacy of pamidronate on BMD with no association with any alteration in graft function during the first year of renal transplantation. Significant heterogeneity precludes the conclusion of the relationship between serum creatinine and pamidronate.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of goserelin in rabbit plasma

A rapid and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry method (LC–ESI-MS/MS) was developed and validated for the determination of goserelin in rabbit plasma. Various parameters affecting plasma sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. Acidified plasma samples were applied to Oasis^® HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100 μL mobile phase A. The separation was achieved on a Capcell-Pak C18 (2.0 mm × 150 mm, 5 μm, AQ type) column with a gradient elution of solvent A (0.05% acetic acid in deionized water/acetonitrile = 85/15; v/v) and solvent B (acetonitrile) at a flow rate of 250 μL/min. The LC–MS/MS system was equipped with an electrospray ion source operating in positive ion mode. Multiple-reaction monitoring (MRM) of the precursor–product ion transitions consisted of m/z 635.7 → m/z 607.5 for goserelin and m/z 424.0 → m/z 292.1 for cephapirin (internal standard). The proposed method was validated by assessing specificity, linearity, limit of quantification (LOQ), intra- and inter-day precision and accuracy, recovery, and stability. Linear calibration curves were obtained in the concentration range of 0.1–20 ng/mL (the correlation coefficients were above 0.99). The LOQ of the method was 0.1 ng/mL. Results obtained from the validation study of goserelin showed good accuracy and precision at concentrations of 0.1, 1, 5, 10, and 20 ng/mL. The validated method was successfully applied to a pharmacokinetic study of goserelin after a single subcutaneous injection of 3.6 mg of goserelin in healthy white rabbits.     

Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes

A high-performance liquid chromatographic assay was developed for the quantitation of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. These compounds along with phenacetin, the internal standard, were extracted from incubation mixtures using ether extraction. The extracts were analyzed on a Brownlee Spheri-5 C₈ column with a mobile-phase of acetonitrile–0.5% phosphoric acid (30:70, v/v). The assay utilized UV detection at 287 nm which provided sensitivity and specificity to simultaneously quantify chlorzoxazone and 6-hydroxychlorzoxazone from liver microsomal samples at amounts of 10 ng and greater. The mean correlation coefficient of the standard curves for 6-hydroxychlorzoxazone and chlorzoxazone was 0.998 and 0.993, respectively, over the range of 25–400 ng, and the regression curves were found to be linear at least through 1600 ng. All components eluted within 7 min, resulting in a total analysis time of 8 min. The inter-day and intra-day coefficients of variation were <7 and <3%, respectively. This method provides a rapid, sensitive and cost-effective assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomal incubations.     

A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) has been developed and validated. The method involved liquid–liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C₁₈ column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1–500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4–99.5% and 89.4–100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 μL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague–Dawley (SD) rats.     

Determination of TJ0711 hydrochloride in rat plasma by high performance liquid chromatography with fluorescence detection and its application to pharmacokinetics

In the present study, a simple and sensitive high performance liquid chromatography with fluorescence detection (HPLC-FD) method was developed to determine TJ0711 hydrochloride, a novel α- and β-receptor blocker. TJ0711 hydrochloride and verapamil hydrochloride (the internal standard) were separated on Knauer Eurospher C₁₈ (250 mm × 4.0 mm i.d., 5 μm) column at 50 °C. The mobile phase was methanol:perchloric acid (12 nM, aq) (56:44, v:v), with a flow rate of 1.0 mL/min. The wavelengths of FD were set at 246 nm for excitation and 300 nm for emission. For plasma samples of rats, the analytes were extracted with acetic ether from alkalinized plasma, and then back-extracted into 10 mM dilute sulfuric acid. The linearity was over a concentration range of 20–10,000 ng/mL. The intra- and inter-day precisions referred by relative standard deviation were less than 2.0% and 4.3%, respectively. The mean analytical recoveries of TJ0711 hydrochloride at different concentrations (50, 1000 and 8000 ng/mL) ranged from 88.3% to 92.9%. The lower limit of quantification (LLOQ) was 20 ng/mL. Finally, this method was successfully applied to the estimation of pharmacokinetic parameters of TJ0711 hydrochloride after intravenous doses of 4, 8 and 16 mg/kg in rats.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

Determination of the anti-platelet-activating factor BN-50727 and its metabolites in human plasma by high-performance liquid chromatography-solid-phase extraction

A sensitive and selective HPLC-solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human plasma. The procedure consisted of an automated solid-phase extraction of the drug and metabolites on disposable propylcarboxylic acid cartridges, followed by on-line chromatographic separation. The method was linear from 3.75 to 2400 ng/ml and the limit of quantitation for BN-50727 in plasma samples was 3.75 ng/ml. The within-run precision of the method, expressed as relative standard deviation, ranged from 2.1 to 8.1%. The accuracy, expressed as relative error, ranged from −3.5 to 4.0%. For the main metabolite, the O-demetthylated BN-50727 product, the method was linear from 7.5 to 2400 ng/ml and the limit of quantitation in plasma was 7.5 ng/ml. The within-run precision ranged from 2.1 to 11.0% and the accuracy from −5.3 to 1.1%. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human plasma and also of its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human plasma after administration of single and multiple doses.     

Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid—liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5–2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Sensitive determination of methomyl in blood using gas chromatography–mass spectrometry as its oxime tert.-butyldimethylsilyl derivative

A sensitive, selective and reliable method was developed to determine methomyl {methyl-N-[(methylcarbamoyl)oxy]thioacetimidate}, a carbamate insecticide in human blood, using gas chromatography–mass spectrometry. Dimethylglyoxime served as an internal standard (I.S.). Methomyl in the blood was converted to its oxime form by sodium hydroxide. The solution made acidic with hydrochloric acid was poured into a column packed with Extrelut. Methomyloxime and I.S. were eluted from the column with a mixture of dichloromethane–ethyl acetate–chloroform (65:25:10), transformed to tert.-butyldimethylsilyl derivatives, and analyzed by gas chromatography–mass spectrometry in the electron impact mode. The calibration curves were linear in the concentration range from 1 ng/g to 100 ng/g and 100 ng/g to at least 5000 ng/g. The lower limit of detection was 0.5 ng/g. The absolute recoveries were 72–93% and within-day coefficients of variation were 3.1–5.6% at blood concentrations of 10 and 1000 ng/g. Two practical forensic applications are described.