Determination of Evans blue as a blood–brain barrier integrity tracer in plasma and brain tissue by UHPLC/UV method

Blood–brain barrier changes are an integral part of many neurodegenerative diseases. Evans blue is an intravital dye that binds to albumin and can therefore be used to monitor extravasation of this plasma protein across blood–brain barrier in animal models of neurodegeneration. To monitor extravasation of albumin across blood–brain barrier, we developed and validated an ultrahigh-performance liquid chromatography method for the analysis of Evans blue in rat plasma and brain samples. Analyte was separated on ACQUITY UPLC BEH C18 column (2.1?mm?×?50?mm) using a 5-min gradient run and detected by a UV detector. The limits of quantification (LOQ) were 10?µg/mL in plasma and 0.5?µg/g in brain samples. The limits of detection (LOD) were 1?µg/mL in plasma and 0.015?µg/g in brain samples. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 91–105%. The inter-day precision was in the range of 1.3–8%. The benefits of using UPLC are selectivity, short analysis period, and thus, a very good sample throughput. Using this method, we analyzed albumin extravasation across blood–brain barrier in transgenic rat model for tauopathy SHR-72 and age-matched control animals.     

Rapid and Highly Sensitive HPLC and TLC Methods for Quantitation of Amlodipine Besilate and Valsartan in Bulk Powder and in Pharmaceutical Dosage Forms and in Human Plasma

Two simple, sensitive, and specific high-performance liquid chromatography and thin-layer chromatography methods were developed for the simultaneous estimation of Amlodipine besilate (AM) and Valsartan (VL). Separation by HPLC was achieved using a xTerra C18 column and methanol /acetonitrile /water/ 0.05% triethylamine in a ratio 40:20:30:10 by volume as mobile phase, pH was adjusted to 3 ± 0.1 with o-phosphoric acid. The flow rate was 1.2 mL min^?1. The linearity range was 0.2 to 2 µg mL^?1 for amlodipine besilate and 0.4 to 4 µg mL^?1 for Valsartan with a mean percentage recovery of 99.59 ± 0.523% and 100.61 ± 0.400% for amlodipine besilate and valsartan, respectively. The TLC method used silica gel 60 F₂₅₄ plates; the optimized mobile phase was ethyl acetate/ methanol / ammonium hydroxide (55:45:5 by volume). Quantitatively, the spots were scanned densitometrically at 237 nm. The range was 0.5–4.0 µg spot^?1 for amlodipine besilate and 2.0–12.0 µg spot^?1 for valsartan. The mean percentages recovery was 99.80 ± 0.451% and 100.61 ± 0.363% for amlodipine besilate and valsartan, respectively. The HPLC method was found to be simple, selective, precise, and reproducible for the estimation of both drugs from spiked human plasma.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

Validated assay for the simultaneous quantification of total vincristine and actinomycin‐D concentrations in human EDTA plasma and of vincristine concentrations in human plasma ultrafiltrate by high‐performance liquid chromatography coupled with tandem mass spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin‐D concentrations in human plasma and an assay for the determination of unbound vincristine are presented. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and heated electrospray ionization (H‐ESI) were tested as ionization interfaces. For reasons of robustness ESI was chosen followed by tandem mass spectrometry (ESI‐MS/MS). For the plasma assay a 30 µL aliquot was protein precipitated with acetonitrile/methanol (50:50, v/v) containing the internal standard vinorelbine and 10 µL volumes were injected onto the HPLC system. To determine unbound vincristine, ultrafiltrate was produced from plasma using 30 kDa centrifugal filter units. The plasma ultrafiltrate was mixed with methanol (50:50, v/v), internal standard vinorelbine was added and 20 µL aliquots were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using 1 mM ammonium acetate/acetonitrile (30:70, v/v) adjusted to pH 10.5 with ammonia, run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies in plasma vincristine from 0.25 to 100 ng/mL and actinomycin‐D from 0.5 to 250 ng/mL using plasma sample volumes of only 30 µL. Vincristine in plasma ultrafiltrate can be quantified from 1 to 100 ng/mL. Validation results demonstrate that vincristine and actinomycin‐D can be accurately and precisely quantified in human plasma and plasma ultrafiltrate with the presented methods. The assays are now in use to support clinical pharmacological studies in children treated with vincristine and actinomycin‐D. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Cefalothin and Cefazolin in Human Plasma,Urine and Peritoneal Dialysate by UHPLC‐MS/MS: application to a pilot pharmacokinetic study in humans

An ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) method for the analysis of cefazolin and cefalothin in human plasma (total and unbound), urine and peritoneal dialysate has been developed and validated. Total plasma concentrations are measured following protein precipitation and are suitable for the concentration range of 1–500 µg/mL. Unbound concentrations are measured from ultra‐filtered plasma acquired using Centrifree^® devices and are suitable for the concentration range of 0.1–500 µg/mL for cefazolin and 1–500 µg/mL for cefalothin. The urine method is suitable for a concentration range of 0.1–20 mg/mL for cefazolin and 0.2–20 mg/mL for cefalothin. Peritoneal dialysate concentrations are measured using direct injection, and are suitable for the concentration range of 0.2–100 µg/mL for both cefazolin and cefalothin. The cefazolin and cefalothin plasma (total and unbound), urine and peritoneal dialysate results are reported for recovery, inter‐assay precision and accuracy, and the lower limit of quantification, linearity, stability and matrix effects, with all results meeting acceptance criteria. The method was used successfully in a pilot pharmacokinetic study with patients with peritoneal dialysis‐associated peritonitis, receiving either intraperitoneal cefazolin or cefalothin. Copyright © 2015 John Wiley & Sons, Ltd.     

Ionic liquids dispersive liquid–liquid microextraction and high‐performance liquid chromatographic determination of irbesartan and valsartan in human urine

An environmentally friendly ionic liquids dispersive liquid–liquid microextraction (IL‐DLLME) method coupled with high‐performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed‐phase C₁₈ column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL‐DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL‐DLLME‐HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a Monoclonal Antibody-Based ELISA for the Detection of Sulfaquinoxaline in Chicken Tissues and Serum

Abstract

A monoclonal antibody (Mab) was produced by using sulfaquinoxaline-human serum albumin (SQX-HSA) conjugate as immunogen. The anti-SQX Mab exhibited negligible cross reactivity with other commonly used sulfonamides. Using this Mab, a competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to detect SQX in chicken tissues and serum. The ciELISA showed a 50% inhibition (IC₅₀) value of 2.60 ng/mL. The recoveries of SQX from spiked chicken muscle, liver, and serum at levels of 5–50 µg/kg were 82.6–96.5%, 75.3–94.5%, and 69.7–89.3%, respectively. The coefficient variations (CVs) were 6.22–7.17%, 4.9–8.9%, and 1.20–10.15%, respectively. Detection limits were 1.29 µg/kg in muscle, 1.32 µg/kg in liver, and 2.44 µg/kg in serum.     

A GC-SIM-MS Method for the Determination of Butylidenephthalide in Rat Plasma and Tissue: Application to the Pharmacokinetic and Tissue Distribution Study

Abstract

Butylidenephthalide is one of the major active components isolated from Rhizoma Chuanxiong. This paper describes a simple, rapid, specific and sensitive method for the quantification of butylidenephthalide in rat plasma and tissue distribution using a liquid-liquid extraction procedure followed by capillary gas chromatography-selected ion monitoring mode-mass spectrometry (GC-SIM-MS) analysis. The calibration curves were linear over the concentration ranging from 0.02–10.0 µg/mL (r > 0.99) for plasma samples and 0.18–7.25 µg/g (r > 0.99) for the tissue samples. The limit of quantification (LOQ) was 1.0 ng/mL or 1.0 ng/g (ten times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.39–2.98% and 2.97–4.26%, respectively. The methods of recovery for all samples were greater than 80% at the low, medium, and high concentrations. The method has been successfully applied to a pharmacokinetics study in rats after an oral administration of Butylidenephthalide with a dose of 20.0 mg/kg. The main pharmacokinetic parameters obtained were T _max  = (0.22 ± 0.06) h, C _max = (3 ± 1) µg/mL, AUC = (32 ± 6) h?µg/mL, and K _a  = (8.5 ± 0.8)/h. The results showed that the butylidenephthalide was easily absorbed. The concentrations of butylidenephthalide in rat kidney, lung, heart, and cerebellum were higher than those in other organs. To determine free fraction in serum, samples were filtered using ultrafiltration membranes with a molecular weight cut-off of 10,000 Da and extracted using liquid-liquid extraction. The extracts were evaporated and analyzed by GC-MS. The protein binding in rat plasma, human plasma, and human serum albumin were 83 ± 4%, 94 ± 3%, and 89 ± 3%, respectively.     

Electrochemical Magnetic Immunosensors for the Determination of Ceruloplasmin

Electrochemical immunosensors for ceruloplasmin (Cp) are reported for the first time. Two configurations involving magnetic beads (MBs) functionalized with Protein A or Streptavidin for immobilization of Cp antibodies were compared, using competitive immunoassay with synthesized alkaline phosphatase‐Cp conjugate. Upon capturing MBs‐immunoconjugates onto screen‐printed carbon electrodes, quantification of Cp was accomplished by DPV measurement of 1‐naphthol generated after 1‐naphthylphosphate addition. Linear ranges of calibration curves and detection limits were 0.1–1000 µg/mL and 0.040 µg/mL (Protein A‐MBs), and 0.025–20 µg/mL and 0.018 µg/mL (Strept‐MBs). Good results were obtained in the determination of Cp in spiked human serum samples.     

Micellar liquid chromatographic method for the determination of ephedrine and pseudoephedrine in human serum by direct inject of the sample with simple dilution

A micellar high-performance liquid chromatographic method was developed to simultaneously determine ephedrine and pseudoephedrine in human serum. The serum sample pretreatment was a simple dilution in a micellar solution, filtration, and direct injection, thus avoiding time-consuming and tedious steps. Hence, there is no need to use an internal standard. The serum samples were analyzed using a mobile phase containing 1.50?×?10^?1?mol/L sodium dodecyl sulfate and 0.02?mol/L sodium dihydrogen phosphate with 7.5% (v/v) 1-propanol at pH 3.0, running at 1.0?mL/min by an Inertsil C18 (150?×?4.6?mm, 5?µm) column at 30°C. The UV wavelength was set at 210?nm. The developed method was validated by linearity (r?>?0.9990) and intra- and inter-day precisions of ephedrine and pseudoephedrine (relative standard deviation; RSD%, 0.04–10.40, and RSD %, 0.30–10.25, respectively), LODs for ephedrine and for pseudoephedrine was 2.63 and 2.70?µg/mL, respectively; lower limit of quantification for ephedrine and for pseudoephedrine was 4.38 and 4.51?µg/mL, respectively. Finally, the proposed method was applied to investigate ephedrine and pseudoephedrine in real human serum samples after oral administration of Kechuanning Koufye including Ephedra herb. It is environmentally friendly, easy-to-handle, and feasible method for routine analysis in clinical laboratory.     

Study on the interactions of sulfonylurea antidiabetic drugs with normal and glycated human serum albumin by capillary electrophoresis‐frontal analysis

Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the body's systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis‐frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis‐frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and automation.     

Effects of the Interaction of Rifamycin SV with Serum Albumins on the Resonance Rayleigh Scattering Spectra and Their Analytical Application

In pH 4.5–4.8 Britton‐Robinson buffer solution, rifamycin SV (i.e. rifamycin sodium) can react with serum albumin such as human serum albumin (HSA) and bovine serum albumin (BSA) to form macromolecular complexes by electrostatic attraction and hydrophobic force. As a result, the resonance Rayleigh scattering (RRS) of the drug was enhanced remarkably and the RRS peaks were at 374 and 552 nm. The enhancement of RRS (ΔI) is directly proportional to the concentration of HSA or BSA. The linear ranges and the detection limits are 0.03–6.0 µg/mL and 9.0 ng/mL for HSA, and 0.01–8.0 µg/mL and 2.0 ng/mL for BSA, respectively. In this work, a sensitive, selective, simple and fast method for the determination of trace amounts of serum albumin by RRS technique has been developed, which was applied to the determination of serum albumin in the synthesized samples and human urine samples with satisfactory results.     

Liquid Chromatographic Analysis of Cephalexin in Human Plasma by Fluorescence Detection of the 9-Fluorenylmethyl Chloroformate Derivative

Abstract

A simple and sensitive precolumn derivatization method for the determination of cephalexin in human plasma has been developed. Cephalexin was derived with 9-fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer (5 mM, pH 8.5) for 15 min at 25°C. Optimal conditions for the derivatization were described. The derivative was chromatographed on an XDB-C₁₈ column with water–acetonitrile (10:90, v/v) as mobile phase at a flow rate of 1.0 mL/min. The fluorescence excitation and emission wavelengths were 268 nm and 314 nm, respectively. The standard curve in spiked plasma was linear over the range of 0.0234–58.5 µg/mL; the detection limit (signal-to-noise ratio = 3; injection volume, 10 µL) was about 0.014 µg/mL. The performance of analysis was studied, and the validated method showed excellent performance in terms of selectivity, sensitivity, precision, and accuracy.     

Simultaneous Quantification of Benazepril, Gliclazide and Valsartan in Human Plasma by LC–MS–MS and Application for Rapidly Measuring Protein Binding Interaction between Rhein and These Three Drugs

For new drug candidates with high protein binding in the treatment of diabetic nephropathy, their influence on the protein bindings of angiotensin-converting enzyme inhibitors, sulfonylurea drugs, and angiotensin receptor blockers should be predicted to prevent side effects. To provide an efficient tool for this study, a sensitive and rapid LC–MS–MS method was developed for the simultaneous quantification of representative drugs, benazepril, gliclazide and valsartan in human plasma. Chromatographic separation was performed on a Shim-pack VP-ODS C18 column (250 × 2.0 mm i.d., 5 μm) using methanol–0.05% formic acid (90:10, v/v) as mobile phase. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in SRM mode. Lower limits of quantification were 2, 2 and 20 ng^?1 mL for benazepril, gliclazide and valsartan with 0.1 mL plasma sample. The method fulfills the precision, accuracy, linearity, sensitivity, selectivity requirements to quantify the three drugs and has been successfully used to studying protein binding of benazepril, gliclazide and valsartan in the presence of rhein.     

Cloud Point Extraction of Serum Albumin and Its Spectrophotometric Determination in Serum Samples

Abstract

A new method based on the cloud point extraction (CPE) separation and ultraviolet spectrometry determination was proposed for the determination of albumin. When the system temperature is higher than the cloud point extraction temperature (CPT) of the mixed surfactant of p‐octyl polyethyleneglycolphenyether (Triton X‐100) and sodium dodecyl sulfate (SDS), serum albumin could be extracted into surfactant‐rich phase. The main factors affecting the cloud point extraction were investigated systematically. Under the optimized conditions, the determination limit for serum albumin as low as 0.18 µg/mL was obtained by preconcentrating a 10 mL sample solution, and the relative standard deviation (n=10, c=40.0 µg/mL) was 3.77%. The proposed method was applied to the determination of albumin in serum samples. The results obtained by this method were in good agreement with coomassie brilliant blue (CBB).     

Simultaneous determination of methotrexate and metoclopramide in physiological fluids using RP-HPLC with ultra-violet detection; application in evaluation of polymeric nanoparticles

Methotrexate (MTX) is an anticancer drug while metoclopramide (MCP) is an antiemetic agent. Both the drugs are commonly coprescribed to avoid the emesis caused by anticancer drug. In this study, a novel, rapid, sensitive, and cost-effective reverse-phase high-performance liquid chromatography method was developed and validated for simultaneous determination of the methotrexate and metoclopramide in biological and pharmaceutical samples using sparfloxacin as internal standard. The analytes were separated on a Kromasil 100-5C18 RP (250?×?4.6?mm, 5?µm) column, methanol, and 0.05% trifloroacetic acid (36:64?v/v) as mobile phase with a flow rate of 1?mL/min, detection wavelength of 290?nm, and column oven temperature at 40°C. Both the analytes were extracted from physiological fluids (bovine aqueous humor, vitreous humor, and human plasma) using mixture of methanol and 10% perchloric acid (50:50 v/v). The method was linear over the concentration range of 0.025–1.0?µg/mL for methotrexate and 0.030–1.0?µg/mL for metoclopramide. The % recovery from human plasma was 98.57 and 96.74% for MTX and MCP, respectively, while from aqueous humor and vitreous humor was 95.84 and 98.51% for MTX.

The developed method was applied for in vitro release of MTX from polymeric nanoparticles and can be applied for analysis of pharmaceutical and biological samples containing both the drugs.     

毛细管电泳-迎头分析法测定血清或血浆中氯氮平的游离浓度

采用毛细管电泳-迎头分析模式体外实验测定了人血清白蛋白溶液、人血浆、兔血清和血浆样品溶液中游离氯氮平的浓度．根据前沿峰的峰高直接测定了样品溶液中的游离氯氮平浓度,并与传统超滤法进行了比较．通过考察施加的电压和运行缓冲液的组成对氯氮平电泳峰平台形成的影响确定了最优分离条件．讨论了氯氮平蛋白结合作用的选择性．     

A novel liquid chromatography-fluorescence method for the determination of delafloxacin in human plasma

Delafloxacin is a novel fluoroquinolone antibiotic that was approved by the European Medicine Agency to treat bacterial infections of the skin and underlying tissues, and community-acquired pneumonia. Despite being in the market since 2019 in the European Union, there is no published liquid chromatography-fluorescence method for delafloxacin quantification in biological samples. A novel, rapid, and sensitive high-performance liquid chromatographic method was developed to determine delafloxacin in human plasma using its native fluorescence. Plasma delafloxacin concentrations were determined by reverse-phase chromatography with fluorescence detection at 405/450 nm of excitation/emission wavelengths. Delafloxacin was separated on a Kromasil C18 column 250 × 4.6 mm id, 5 µm using isocratic elution. The mobile phase was a mixture of 0.05% trifluoroacetic acid/acetonitrile (52/48). Retention times were 5.4 and 11.6 min for delafloxacin and valsartan (internal standard), respectively. Regression calibration curves were linear over the range of 0.1–2.5 µg/mL. The lower limit of detection was 0.05 µg/mL, and the lower limit of quantification was 0.1 µg/mL. Accuracy and precision were always <11%, and the limit of quantification was <16%. Mean recovery was 98.3%. This method can be applied to determine delafloxacin in human plasma and could be useful to perform pharmacokinetic studies.     

A novel HPLC-UV method for determining alendronate in rat plasma through precolumn derivatization with phenyl isothiocyanate: Application to pharmacokinetics

The determination of alendronate (ALE) in biofluids using a low-cost instrument is potentially useful in preclinical pharmacokinetic studies. This study developed and validated a high-performance liquid chromatography with ultraviolet method for ALE determination in rat plasma using precolumn derivatization with phenyl isothiocyanate (PITC). Inhibiting compounds in the samples were first eliminated using solid-phase extraction. ALE in the sample was subsequently allowed to react with PITC to form a phenylthiocarbamoyl derivative for further analysis. The assay was linear within the concentration range of 0.29–25.0?µg/mL. The precision and accuracy were less than 3.9% and 98.0?±?3.9%, respectively. The limits of detection and quantification were 0.08 and 0.20?µg/mL, respectively. The method was successfully used to evaluate the pharmacokinetic parameters of ALE in rats following a single oral administration (30.0?mg/kg). The results show that the peak plasma ALE concentration is 0.69?±?0.18?µg/mL. The area under the plasma concentration–time curve value of ALE was 2.14?±?0.68?µg/mL hr. This method can suitably evaluate the bioavailabilities of different ALE dosage forms in preclinical pharmacokinetic studies.     

Chromatographic analysis of ledipasvir and sofosbuvir: New treatment for chronic hepatitis C infection with application to human plasma

Sofosbuvir (SOF) and ledipasvir (LED) are recently approved and coformulated as directly acting antiviral agents used for treatment of hepatitis C virus (HCV). A reversed phase high performance liquid chromatography - diode array detector (RP-HPLC/DAD) method was developed and validated for the first time for the analysis of newly formulated anti-HCV combination, in pure form, pharmaceutical formulation and in human plasma. In the developed method, separation was performed on Zorbax® Eclipse C18 column using a gradient mixture of acetonitrile–water as a mobile phase and scanning was performed at 260?nm (for SOF) and 330?nm (for LED). The two drugs were completely separated from each other and from plasma, where plasma peak appeared at 2.76?±?0.05?min, SOF at 4.25?±?0.05, and LED at 7.35?±?0.05. The developed method showed high sensitivity, the drugs showed linearity in the range of 1–45?µg/mL for both pure form and spiked human plasma. Three freeze–thaw cycles were performed separately at two different temperatures, ?8 and ?20°C. No significant loss of the studied drugs were observed during repeated thawing and freezing. Validation parameters such as accuracy, precision, robustness, and ruggedness were tested in compliance with USP recommendations, where acceptable results were obtained. Applying to pharmaceutical formulation showed no interference from tablet excipients.