Current Pharmaceutical Analysis - Volume 14, Issue 3, 2018

Background: Performance enhancement substances and methods other than exercise training and physical conditioning have become a major problem in athletic competitions. Over the last few years, there has been an increase in the number of these doping approaches used by some athletes, including pharmaceuticals, slightly modified endogenous compounds and also blood transfusions. In order to control and prevent these doping practices by athletes, World Anti-Doping Agency has stipulated several guidelines and approved various methods on the basis of reproducibility, sensitivity and adaptability. The number, design and type of doping substances are increasing on daily basis necessitating the rapid development of analytical methods to detect these substances and to prevent doping. Objective: In this review, we address the various methodological developments in the last few years to track down the novel doping substances as well as doping methods. Results: There have been significant advances in the area of mass spectroscopy and the associated detection devices to measure small quantities of test substance or their metabolites in body tissues and fluids. Some of the doping substances have short biological half-life but leave imprints of their action in the form of altered gene expression, protein expression or metabolism, which can be detected by OMICs technologies. Conclusion: The rapid advance in biological instrumentation and our understanding of the molecular basis of the actions of doping substances have paved the way to enforce ‘true play’ in athletic competitions. But this is an incessant and a continuous process as long as the doping practices continue.

Background: A growing attention was paid on the antitumor activities of Pulsatilla chinensis (Bunge) Regel. For better pharmacological elucidation and further safety evaluation, we characterized tissue distribution of five active pulchinenosides for solubilization formulations of Pulsatilla chinensis saponins in a tumour-bearing mice model. Hypothesis/Purpose: The active ingredients level at the site of action tissues is the relevant measure of drug effect. Especially for antitumor drug formulations, their biodistributions are crucial to investigating the target tissues and potential accumulative organs. Study Design: Tissue distributions of the various water-solution formulations delivering saponins in vivo were taken for the first time. In addition, to evaluate probability of tissues accumulation, active components levels in various organs were investigated after a long-term administration. Methods: The extract of PRS was prepared for PRS-Na, PRS-HPβCD, PRS-O/W, PRS-silica, PRS-Na- HPβCD, respectively. Heterotopic transplanted liver tumor model mice were adopted. The PRS levels of tissues were determined by HPLC-MS/MS method. Results: PRS-Na was a suitable oral formulation for saponins treating cancer. Lung was potential target tissue for PRS-O/W, while liver for PRS- HPβCD, and PRS-Na-HPβCD. Among all the designed formulations, PRS-micronized silica was most unsuitable for saponins treating liver cancer. Besides, low dosage (80mg/kg) daily administrated to mice not showed significant tissue accumulation. However, fat storage would be noticed in high dosage (300mg/kg) of PRS-Na, PRS- HPβCD, and lung of PRS- micronized silica formulation. Conclusion: Disposition into target tissues plays an important role for pharmacological effects, especially for natural compounds. Based on the results of pharmacokinetics and distribution results, PRS-Na was a suitable oral formulation for saponins treating cancer. Fat accumulation for long-term administration was not ignored for saponins formulations.

Background: Spironolactone, an aldosterone antagonist and a diuretic, is unstable in aqueous solution. The stability-indicating method has not been reported for use in the assessment of spironolactone stability in the developed oral liquids. Objective: To develop and validate a stability-indicating HPLC method for determination of spironolactone in parabens-preserved oral liquids formulated with and without hydroxypropyl-β-cyclodextrin. Methods and Results: A reversed phase HPLC method was developed. Baseline separation was achieved on a C18 column at room temperature (25°C) with a mobile phase consisting of 10 mM ammonium acetate buffer pH 4 and methanol (42:58, v/v) at a flow rate of 1.0 mL/min. Detection and peak purity assessments were performed by photodiode array detector at 238 nm along with the 200 through 400 nm scan mode. The method was validated over the range of 40% to 120% labeled amount of spironolactone in oral liquids. It was highly selective, accurate and precise. It provided chromatograms with good peak efficiency and acceptable resolution in approximately 9 min. Parabens, parabens degradation product, spironolactone and spironolactone degradation products were all well resolved. Peak purity analysis also demonstrated that the spironolactone peak was pure. The accuracy was in the range of 98.50-101.5%. The within-run and between-run relative standard deviations were not greater than 2%. The calibration curve was linear over the concentration range of 10.0-60.0 μg/mL (r² > 0.9999). This developed method was successfully applied to the stability study of spironolactone in oral liquids. Conclusion: The developed method was valid and applicable for stability study of spironolactone in the oral liquids.

Background: Recombinant human interleukin-11 (rhIL-11) is a cytokine produced by recombinant DNA technology and used clinically to treat chemotherapy-induced thrombocytopenia. A Size Exclusion Liquid Chromatography (SE-LC) method was validated to assess the content/potency of the rhIL-11. Methods: A BioSep-SEC-S2000 column (300 mm 7.8 mm i.d.) maintained at 30°C was used. The mobile phase was composed of 0.02 M 2-(N-morpholino)ethanesulfonic acid and 0.5 M sodium chloride buffer, pH 6.0, run isocratically at a flow rate of 0.9 mL min-1, with detection by Photodiode Array (PDA) detector set at 220 nm. Results: Chromatographic separation was obtained with a retention time of 8.12 min, and the stabilityindicating capability, and specificity was demonstrated. The method was linear over the concentration range of 1.0–200 μg mL-1 (r²= 0.9996) and the Detection Limit (DL) and Quantitation Limit (QL) were 0.23 μg mL-1 and 1.0 μg mL-1, respectively. The accuracy was 99.81% with bias lower than 0.43%. Moreover, method validation showed acceptable results for precision and robustness. Conclusion: The method was applied to the assessment of rhIL-11 and Higher Molecular Weight forms (HMW) in biopharmaceutical formulations, and the results of content/potencies were correlated to those of a validated Reversed-Phase Liquid Chromatography (RP-LC) and a Capillary Zone Electrophoresis (CZE) methods, showing non-significant differences (p> 0.05). The proposed method and the correlation studies contribute to monitor stability, improve the quality control and ensure the batch-to-batch consistency of the biotherapeutics.

Objective: The present study describes the development and validation of a simple highthroughput method for the fluorometric determination of besifloxacin HCl in 96-microwell plates. Methods: The proposed method is based on the reaction between besifloxacin HCl and fluorescamine in borate buffer solution of pH 8.5 giving a highly fluorescent derivative that was developed simultaneously and measured at 471 nm after excitation at 353 nm. The different experimental conditions affecting the fluorescence reaction were carefully investigated and optimized. Results: The method showed linearity over the concentration range of 200-1000 ng/mL with low Limits Of Detection (LOD) and Quantitation (LOQ) of 8.47 ng/mL and 28.24 ng/mL, respectively. The Relative Standard Deviation (% RSD) and Relative Error (% RE) values did not exceed 3 % for precision and accuracy respectively. The developed high-throughput method was successfully applied for the determination of besifloxacin HCl in simulated tears and eye drops. Conclusion: The capability of the method for measuring micro-volume samples makes it convenient for handling a very large number of samples simultaneously. In addition, using micro-well plates can offer a safer and economic method for the determination of besifloxacin HCl in pharmaceutical dosage forms.

Introduction: The majority of analytical methods used currently to quantify Vitamin B6 are time-consuming, costly, non-environment friendly and destructive with respect to the analyzed material. Objective: The objective of this study was to elaborate a novel method of vitamin B6 quantification in commercial pharmaceutical preparations using Near Infrared Spectroscopy (NIRS). Methods: Powdered tablets and calibration samples containing pyridoxine hydrochloride and microcrystalline cellulose or lactose were employed. The raw NIRS spectra of calibration samples and tablets were transformed by Multiplicative Scatter Correction (MSC) or by second derivative normalization and used to generate the appropriate calibration plots. Alternatively, determination of pyridoxine hydrochloride was performed by Principle Component Regression (PCR) for powdered tablets and calibration sets of samples. The relationship was established between the Principal Component PC1 calculated for the selected ranges of pretreated NIRS reflectance spectra of calibration standards and the contents of pyridoxine hydrochloride. Results: The samples of 3 different preparations containing 10 or 50 mg of pyridoxine hydrochloride that differed in composition of the excipients were analyzed. Depending on the composition of the formulation used and the selected wavelength range, the best recoveries for tested preparations were between 100-102%. Conclusion: In this study we have proved that quantification of hydrochloride pyridoxine in solid pharmaceutical preparations by Near Infrared Reflectance Spectroscopy may be achieved with high precision and accuracy. Our method is cheap, rapid, environmentally-friendly and may be used for routine analyzes in the Quality Control laboratory.

Background: A simple, sensitive and precise spectrodensitometric method has been developed and validated for simultaneous determination of sofosbuvir, ribavirin and saxagliptin in their pure and pharmaceutical dosage forms. Methods: The method employed TLC plates precoated with silica gel G 60 F254 as the stationary phase. The mobile phase consisting of acetonitrile-water (80:20, v/v) was used to give compact bands for all the studied drugs at 228 nm. They were resolved with retardation factor (Rf) values of 0.71, 0.36 and 0.21 for sofosbuvir, ribavirin and saxagliptin respectively. Results: The linearity ranges were 400 – 10000 ng/band for all the studied drugs. Limits of detection were 124.78, 124.31 and 128.29 and limits of quantitation were 378.13, 376.71, 388.77 ng/band for sofosbuvir, ribavirin and saxagliptin, respectively. Correlation coefficient (r) values were 0.9993, 0.9995 and 0.9991 for sofosbuvir, ribavirin and saxagliptin, respectively. Conclusion: The method was validated according to ICH guidelines and has been successfully applied for the determination of the studied drugs in their pure and dosage forms without any interference from the commonly encountered excipients.

Background: Propylthiouracil (PTU)-induced liver injury has been concerned in recent years and the mechanisms are largely unknown. Therefore, establishment of an analytical method for PTU detection in hepatic cells would contribute to the investigation of PTU hepatic injury. Objectives: In this study, a simple and rapid high performance liquid chromatography (HPLC) method for the intracellular determination of PTU concentration in rat liver cells (BRL-3A) was first developed and then validated. Methods and Results: The cell samples were pretreated with methanol to precipitate protein and analyzed by HPLC. The mobile phase was methanol-water (40:60) at a rate of 1.0 mL/min. Luna C18 column was adopted with detection wavelength at 272 nm. Specificity of the assay showed that endogenous components and solvents had no interference to PTU measurement. The linearity of method was demonstrated in the range of 0.0833-0.833 μg/mL with the correlation coefficient of 0.9968. The LOD and LOQ were 0.0580 μg/mL and 0.0833 μg/mL, respectively. The average extraction recovery of PTU from cell lysates was 91.9%. Accuracy of the method was found between 98.7% and 103.0%. The precision of intraday and interday (RSD) was less than 5.6%. Conclusion: This method is simple, accurate, and specific, which is suitable for PTU examination in hepatic cells.

Background: It is highly crucial to clarify the pharmacokinetic mechanism for the active multi-ingredients in Traditional Chinese Medicines so that suggestions could be provided in clinical. Introduction: A novel, rapid and sensitive Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC–MS/MS) method was developed for the simultaneous determination of four compounds (tetrahydropalmatine, danshensu sodium, ferulic acid and rosmarinic acid) after oral administration of Guanmaitong Granule in rat plasma using puerarin as the Internal Standard (IS). Method: Acidified plasma samples were extracted by liquid–liquid extraction method with ethyl acetate, and separated on a Waters ACQUITY UPLC® BEH C18 column (50.1 mm, i.d., 1.7 μm) using a gradient mobile phase system composed of acetonitrile–water containing 0.1% formic acid. The detection was performed in multiple reaction monitoring mode with positive and negative-ion mode. Result: The method was validated over the concentration range of 0.005-20.48 ng/mL for tetrahydropalmatine, 5.4-100 ng/mL for danshensu sodium, 8.8-1000 ng/mL for ferulic acid and 3-60 ng/mL for rosmarinic acid; the Lower Limit Of Quantification (LLOQ) was between 0.005 ng/mL and 8.8 ng/mL for the four analytes. The intra-day and inter-day precisions were all less than 15% and the accuracies were within the range of 85%-115%. Extraction recovery, matrix effect and stability were also satisfactory. Conclusion: The validated method was successfully applied in a pharmacokinetic study in rats after oral administration of Guanmaitong Granule.

Background: An accurate HPLC-fluorescence method for the simultaneous determination of abiraterone acetate (prodrug) and abiraterone (drug) employed in the treatment of prostate cancer, in urine and serum samples of men treated with this prodrug has been proposed. Methods: The developed HPLC-FLD procedure permits the quantification of ABR and AA minimizing laborious and complicated sample preparation procedures. The selectivity of the fluorescence detector avoids the presence of endogenous and exogenous interfering compounds and has enough sensibility for this determination. Results: The method was applied for the analysis of human urines samples of five men and serum samples of two men under treatment of prostate cancer. The excretion urinary in a patient was done during 24 hours. The maximum abiraterone concentration for this patient was 27 ng/mL. Conclusion: This method shows the advantage of being suitable for the analysis of urine and serum samples of patients with prostate cancer who received daily doses of Zytiga.

Background: The determination of NSAIDs (ketoprofen, diclofenac, and ibuprofen) in urine samples provides useful information for assessing their safety, therapeutic effect, and their mechanism of action. Urine samples are characterized by their complexity and low concentration of the target analytes, which make their direct analysis difficult. In this work, the potential of multi-walled carbon nanotubes (MWCNTs) as Solid Bar Microextraction (SBME) adsorbents for extraction and preconcentration of selected drugs from urine samples have been investigated. Methods: Five SBME devices (contains 10 mg of MWCNTs) were placed in 30 mL of adjusted pH 2 water samples and stirred for 40 minutes. After finishing the extraction, the devices were ultrasonicated in 250 μL of methanol for 5 min to desorb the selected drugs and analyzed using HPLC-DAD. Results: The analytical performance of the whole method was evaluated in water samples. Limits of detection and quantitation were in the ranges of (0.36- 0.52) and (1.2- 1.7) μg L-1, respectively, and the Relative Standard Deviation (RSD) < 5.7% over a concentration range of 5-100 μg L-1. Application: The proposed method was successfully applied to the analysis of selected drugs in spiked human urine samples. The absolute recovery obtained by spiking the urine samples at three concentration levels: 5, 50 and 100 μg L-1 of selected NSAIDs were from 48.8% to 53.5%. Conclusion: The proposed SBME using MWCNT as a sorbent material can be a useful alternative sample preparation technique for the routine determination of NSAIDs in urine samples at therapeutic levels with very simple sample pretreatment. Furthermore, the application of the developed SBME method might be extended to study the determination of selected non-steroidal anti-inflammatory drugs in veterinary urine samples.

Background: Levofloxacin (LVX) is one of the fluoroquinolone antibiotics that possess a wide spectrum of bactericidal activity against both Gram-positive and Gram-negative bacteria. Because of its side effects and its potential towards the development of antibiotic resistance, determination of this antibiotic is very important. New methods which have superior properties than the conventional methods are needed in the determination of pharmaceuticals in different samples. Method: An activated pencil graphite electrode (A-PGE) was prepared and its application in the voltammetric determination of LVX in pharmaceutical samples and body fluids was searched. Results: Oxidation peak currents of LVX were increased 40 times in the presence of A-PGE according to bare PGE. The highest voltammetric response of A-PGE towards LVX was achieved at pH value of 4. A wide linear range (0.01 – 2.5 μM) was observed between the oxidation peak currents and concentrations of LVX. Detection limit was calculated as 0.0075 μM. The inter-day (five days) precision of APGE was found to be 3.16%. Interference studies showed that A-PGE successfully discriminated the voltammetric response of LVX from that of many other structurally related substances such as ciprofloxacin, norfloxacin and enoxacin. Prepared A-PGE preserved 87% of its original activity towards LVX after a 2-month storage. Determination of LVX in blood serum, urine and pharmaceuticals was successfully achieved by A-PGE. Conclusion: The prepared A-PGE brings two important advantages. First one is the selective response of A-PGE towards LVX in the presence of other fluoroquinolone antibiotics (ciprofloxacin, enoxacin and norfloxacin). The other is the high sensitivity of A-PGE towards LVX having the detection limit of 0.0075 μM. In addition to the given advantages, its disposable character, low cost, commercial availability and easy preparation of A-PGE make this electrode an important candidate in the fast and accurate determination of LVX.

Background: In this paper, an isocratic reverse phase liquid chromatography method was developed and validated for the determination of curcumin and quinine in nanoformulation using a photodiode array detector. Methods: The optimum chromatographic condition with adequate resolution for curcumin (3.8 minutes, 426 nm) and quinine (8.9 minutes, 232 nm) was achieved when the separation was carried out using a Waters RP-18 (4.6 mm x 300 mm) column employing acetonitrile, methanol and aqueous 0.01 % triethylamine (pH adjusted to 3.0 with phosphoric acid) (45:35:20, v/v/v) as the mobile phase, and flow rate of 1.0 mL min-1. The specificity and stability-indicating capability of the method was verified subjecting reference substance and the formulation to hydrolytic, oxidative, photolytic, and thermal stress conditions. Results: The results obtained from degradation studies demonstrated that the antimalarial nanoformulation proposed was more stable, when compared with the solutions of these drugs in the associated and individual forms. The influence of mobile phase composition, pH, and flow rate on resolution was investigated. The method was validated with respect to linearity, limits of detection and quantitation, precision, and accuracy. The response was linear over a range of 12.0 to 18.0 μg mL-1 (r = 0.999), for both drugs. The limits of detection and quantitation were found to be 1.2 and 3.6 μg mL-1, for curcumin, and 0.6 and 1.8 μg mL-1, for quinine. Conclusion: Further, the proposed method was found to be reproducible and convenient for stabilityindicating analysis of curcumin and quinine-loaded nanoemultion.

Background: Carbohydrates are of great interest for the synthesis of novel ribonucleosides and C-Nucleosides which often show different pharmacological potential including antiinflammatory and antineoplastic characteristics. Introduction: In this research twelve aldopentose derivatives were examined and their chromatographic properties were used to describe their pharmacokinetic profiles. Methods: Thin layer chromatography was performed using three mobile phases: acetone–water (φ = 0.5–0.7 v/v), dioxane–water (φ = 0.5–0.7 v/v) and methanol–water (φ = 0.5–0.7 v/v). Multiple linear regression was performed to examine correlation between lipophilicity and pharmacokinetic descriptors of the examined molecules. Results: Good oral absorption can be expected for all investigated compounds. Moderate volume of distribution indicates low to moderate probability of their accumulation in body tissues. All investigated molecules show good pharmacokinetic characteristics but compounds 2, 3, 5, 6 and 7 demonstrated the best biological potential and biochemical activity such as inhibition of protease and kinase (compound 7) and possibility to be a ligand for GPCR. Conclusion: Among the best candidates authors would emphasize structure 7 as the most promising molecule regarding its pharmacological potential.

Introduction: Piroxicam (PRX) is a widely consumed anti-inflammatory drug, and the assays of its tablets and gels for quality control are mostly performed through High Performance Liquid Chromatography with Ultraviolet Detector (HPLC-UV). Methods: In this regard, an alternate approach, LaFeO3 nanoparticles-modified carbon paste electrode was prepared in order to carry out PRX analysis by using voltammetric approaches. Results: The modified electrode showed better analytical signal. The calibration curve is in the concentration range of 9.00 and 180.00 μmol·L-1 and the limit of detection was obtained as 1.00 μmol·L1 for PRX. The calculated recovery rate was fairly good (99.14%) in comparison with the declared value in the commercial product. Conclusion: As shown by the low overall process cost, performance quickness and analytical results, the proposed method of electroanalysis with the LaFeO3 nanoparticles-modified carbon paste electrode presents a viable and interesting alternative approach to identification and determination of PRX compound in commercial products.

Background: A simple, precise, selective and Reproducible Reversed Phase Ultra-High Performance Liquid Chromatography (RP-UHPLC) method has been developed and validated for the simultaneous determination of two plant constituents, Curcumin (Cur) and Thymoquinone (THQ) in lipid based self-nanoemulsifying formulation and commercial products (Turmeric 500mg capsules and black seed oil 500ml). Method: The chromatographic separation was carried out on a reversed-phase Acquity® BEH C18 column using methanol: 0.25% formic acid solution in water (60:40, v/v) as the mobile phase. The isocratic flow rate was 0.3 ml/min with a rapid run time of 4 min. The UV detections were at 254 nm for THQ and 428 nm for Cur. The method was validated over a concentration range of 100-50,000 ng mL-1 (r²= >0.9999 for both Cur and THQ). Result: The selectivity, specificity, recovery, accuracy and precision were validated for determination of Cur and THQ in lipid based formulation. The lower limits of detection and quantitation of the method were 25 and 75 ng mL-1 for Cur and 35 and 100 ng mL-1 for THQ, respectively. The within and between-day coefficients of variation were less than 5%. The validated method has been successfully applied to quantify both Cur and THQ in self-nanoemulsifying formulation and commercial products. In addition, the proposed method was also applied in ex-vivo (permeability) study using an animal model (rat) to illustrate further the scope and application of the method.

Background: Quercetin (Qur), a potent antioxidant flavonoid have been reported with wide applications in cardiovascular and cerebral ischemic diseases however Qur is prone to degradation in different stress conditions i.e. oxidation, alkaline and acidic medium, photodegradation under UV-light as well as thermal and enzymatic degradation by intestinal bacteria. Objective: The aim of current research is; to study Qur degradation under different stress conditions i.e. oxidation, alkaline and acidic medium and photodegradation under UV-light, to develop and validate a sensitive ultra-high pressure liquid chromatography (UHPLC-MS/MS) method for Qur estimation after different stress conditions as well as in rat brain homogenate and plasma. Method: The estimation study was performed on Waters ACQUITY UPLC^TM with chromatographic conditions set as; column: BEH C-18 with dimensions i.e. 2.1 mm00 mm, 1.7μm particle size, mobile phase: Acetonitrile: 2mM Ammonium Acetate: Formic Acid (90:10:0.01 v/v/v) with a flow rate of 0.20 mL/min. Results: The result showed, transitions for Qur and Internal Standard (IS) Rutin (RT) at m/z 301.0872/151.0309 and 609.2109/299.1203, respectively alongwith a linear dynamic range i.e. 1.00 ng mL-1 to 2000.0 ng mL-1(r² 0.999±0.0001), lower limit of quantification (LLOQ) i.e. 0.054 ng/mL and lower limit of detection (LLOD) with 0.017 ng/mL for Qur. Furthermore, the stability order observed following Qur degradation kinetics was as; alkaline medium (t1/2, 106.28 h-1; t0.9, 17.38 h-1) > acidic medium (t1/2 258.32 h-1; t0.9 40.32 h-1) > oxidative medium (t1/2, 29.32 h-1; t0.9 5.68 h-1). Conclusion: The developed UHPLC-MS/MS method exhibited a successful application in order to study degradation and pharmacokinetic studies in Wistar rat brain homogenate whereby an acceptable precision alongwith adequate sensitivity and accuracy was revealed.

Background: Finasteride is a medically important compound belonging to 4-azasteroids which are widely used in the treatment of benign prostatic hypertrophy. Therefore, there is a need to find a simple, cost effective and sensitive method for the determination of finasteride in commercially available tablet dosage form. Objective: To develop and validate a rapid and simple RP-TLC method combined with densitometry for the quantification of finasteride in tablet dosage form containing 5 mg of active substance alone. Method: Chromatographic analysis was performed on aluminum plates precoated with silica gel 60 RP18F254 using mobile phase consisting of 1,4-dioxane-water in volume composition 35:15. Densitometric scanning was done in the absorbance mode at 212 nm. Validation of proposed RP-TLC method was carried out according to the ICH guidelines. Results: Validation data indicated that the proposed RP-TLC method was accurate and precise with coefficient of variation CV, to be less than 2% in both cases. The method was linear over the range of 1.00÷4.00 μg/spot with correlation coefficient equal to 0.9981. The LOD and LOQ were found to be 0.24 and 0.74 μg/spot, respectively. Assay results of finasteride in marketed tablets using this method were in agreement with label claim and also with pharmacopoeial requirements. Conclusion: A new, simple and economical RP-TLC method with densitometry developed in this study can be found to be suitable for routine analysis of finasteride in simple tablet dosage form containing this active substance.

Introduction: Until date, the methods employed in the quantification of midazolam during sedation and in pre-anesthetic patients are somewhat complex and costly. Hence, the development of simple, practical, precise and reliable method becomes extremely important to measure plasma midazolam levels that could allow its monitoring during sedation, especially in patients who need higher doses of midazolam to achieve deep sedation, a situation which is usually fraught with the risk of increase drug levels and toxicity. Objective: The aim of the present study was to develop a simple and reliable method to monitor plasma levels of midazolam in critically ill pediatric patients. Methods: The plasma levels of midazolam were monitored with 200 μl of plasma during infusion in pediatric patients from Intensive Care Unit using a Pursuit C18 column and UV detection of 220 nm. Results: A linearity ranged between 10-10000 ng/ml of midazolam was obtained with a correlation coefficient of 0.999. The inter and intra-day variation had less than 4% of a coefficient of variation and a mean recovery rate of 99%. Monitoring was carried out in 13 children (from 1-17 years old) at 1, 12 and 24 h post-dose and the median values reached were 437.2, 1908.3 and 4077.6 ng/ml, respectively. It is worth mentioning that some patients received doses 4 times greater than the recommended dose. Conclusion: A simple and reliable HPLC method was developed to monitor midazolam levels in critically ill pediatric patients.

Background: Ethionamide (ETA) is widely used as one of the agents for the treatment of multidrug resistant tuberculosis. Although quality control is an important issue, a fast LC-UV method for the assay and impurity determination of ETA was lacking. So, the aim of this study was to develop such a method to evaluate drug products and follow up dissolution tests of ETA tablets. Methods: Chromatographic separation was achieved using a Hypersil BDS C18 (150 mm.6 mm, 3 μm) column kept at 30 °C. The mobile phase consisted of 300 mL of acetonitrile, 0.14 g of NH4H2PO4 and 700 mL of water pumped at a flow rate of 1.0 mL/min. UV detection was performed at 287 nm. The developed LC method was validated according to the ICH guidelines. Results: Validation results show that the developed LC method is specific, linear, sensitive, precise, accurate and robust. Forced degradation studies revealed that the generated degradation products did not interfere with ETA and known impurities, thus proving the specificity of the developed LC method for the assay of ETA tablets and quantification of impurities. Conclusion: A robust, selective, sensitive and fast LC method has been developed and validated for analysis of ETA and its main impurities in tablets.