RP-HPLC determination of midecamycin and related impurities

Summary A method has been developed for separation and quantitation of midecamycin A₁ and related impurities by high-performance liquid chromatography with evaporative light-scattering detection (ELSD). Chromatographic conditions included use of a Diamonsil C₁₈ column; the mobile phase was 52:48 acetonitrile −0.2 mol L⁻¹ ammonium formate solution (adjusted to pH 7.3 with triethylamine) at a flow rate of 1 mL min⁻¹. The column temperature was 35°C, the shift tube temperature of the ELSD was 105°C, and the gas flow rate of the ELSD was 3.0 L min⁻¹. The response factors of midecamycins in HPLC-ELSD were the same; the linear equation wasy=599292.44x+2868618.04,r=0.9979, the linear range was 5–80 μg,RSD=0.21–1.54%, and theLOD andLOQ were 0.36 and 1.2 μg, respectively. The method was simple, quick, and precise and could be used to determine midecamycin and its related impurities directly.     

Simultaneous RP-LC Determination of Ketorolac and its Piperazinylalkyl Ester Prodrugs

A simple, rapid and selective RP-HPLC method was developed and validated for the determination of ketorolac and five piperazinylalkyl ester prodrugs. A binary isocratic mobile phase composed of a mixture of 65:35 (v/v) 0.02 M phosphate buffer (pH 5.4) and acetonitrile was used on a C₁₈ column (125 × 4 mm, 5 μm). The injection volume was 25 μL and the detection wavelength was 314 nm and the flow rate was 1.5 mL min⁻¹. The method exhibited excellent linearity with R ² of no less than 0.999 and intra-assay and inter-assay precision that were less than the maximum amount allowed according to Horwitz equation. The accuracy was found to be within the allowed ±15%. The limits of detection for the analytes were between 0.060 and 0.220 μg mL⁻¹ and the limits of quantification were between 0.183 and 0.667 μg mL⁻¹. This method was used successfully for the study of the solubility, stability and partition coefficients of piperazinylalkyl ester prodrugs of ketorolac.     

Simultaneous RP-LC Determination of Losartan Potassium, Ramipril, and Hydrochlorothiazide in Pharmaceutical Preparations

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic method has been developed for simultaneous determination of losartan potassium, ramipril, and hydrochlorothiazide. The three drugs were separated on a 150 mm × 4.6 mm i.d., 5 μm particle, Cosmosil C₁₈ column. The mobile phase was 0.025 m sodium perchlorate–acetonitrile, 62:38 (v/v), containing 0.1% heptanesulphonic acid, pH adjusted to 2.85 with orthophosphoric acid, at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. The method was validated for linearity, accuracy, precision, and limit of quantitation. Linearity, accuracy, and precision were acceptable in the ranges 35–65 μg mL⁻¹ for losartan, 1.75–3.25 μg mL⁻¹ for ramipril, and 8.75–16.25 μg mL⁻¹ for hydrochlorothiazide.     

Development and Validation of a Stability-Indicating LC Method for Determination of Ebastine in Tablet and Syrup

A simple stability-indicating reversed-phase liquid chromatographic method with diode-array detection was developed and validated for the quantitative determination of ebastine in tablets and syrup. The LC method was carried out on a C₁₈ column with acetonitrile:phosphoric acid 0.1% pH 3.0 (55:45, v/v) as mobile phase, at a flow rate of 1.2 mL min⁻¹. Ultraviolet detection of ebastine was at 254 nm. A linear response (r = 0.9999) was observed in the range of 10–80 μg mL⁻¹. The RSD values for intra- and inter-day precision studies showed good results (RSD < 2%) and accuracy was greater than 98%. Validation parameters such as specificity and robustness were also determined. The method was found to be stability-indicating and can be applied to quantitative determination of ebastine in tablets and syrup.     

Simultaneous determination of hydrochlorothiazide and losartan potassium in tablets by high-performance low-pressure chromatography using a multi-syringe burette coupled to a monolithic column

This contribution describes use of a separation method based on on-line coupling of a multisyringe flow system with a chromatographic monolithic column for simultaneous determination of hydrochlorothiazide and losartan potassium in tablets. The system comprised a multisyringe module, three low-pressure solenoid valves, a monolithic C₁₈ column (25 mm × 4.6 mm i.d.), and a diode-array detector. The mobile phase was 10 mmol L⁻¹ potassium dihydrogen phosphate (pH 3.1)-acetonitrile-methanol (65:33:2 v/v/v) at a flow rate 0.8 mL min⁻¹. UV detection was carried out at 226 nm. The multi-syringe chromatographic (MSC) method with UV spectrophotometric detection was optimized and validated. Results from validation were very good. The analysis time was about 400 s. The method was found to be applicable to routine analysis of both compounds in tablets. The coupling of the monolithic columns with a multi-syringe flow-injection analysis manifold provides an excellent and inexpensive tool to solve the separation problems without use of HPLC instrumentation.     

Analysis of spectinomycin in fermentation broth by reversed-phase chromatography

A pre-column derivatized high-performance liquid chromatographic (HPLC) method with ultraviolet-visible detection was developed to measure the concentrations of spectinomycin in fermentation broth. Derivatization reagents, 2,4-dinitrophenylhydrazine in acetonitrile (5 mg mL⁻¹) and trifluoroacetic acid in acetonitrile (0.8 mol L⁻¹), were added to an aliquot of the fermentation broth, and the mixture was incubated for 60 min at 70°C. The resulting derivative was separated from other compounds by isocratic elution in a reversed-phase column Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm). Mobile phase consisted of acetonitrile, tetrahydrofuran, and water (φ _r = 40: 35: 25) and the flow rate was 1.0 mL min⁻¹. The detection wavelength was 415 nm. The standard curve for spectinomycin sulfate was linear with correlation coefficients of 0.9997 in the range of 25 μg mL⁻¹ to 600 μg mL⁻¹. The relative standard deviation values ranged from 0.43 % to 2.18 % depending on the concentration of samples. The average recovery was 101.5 %. The limit of detection was 50 ng mL⁻¹.     

LC-ESI-MS Method for the Determination of Mizolastine in Human Plasma

A sensitive and rapid liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the determination of mizolastine in human plasma using dipyridamole as the internal standard (I.S.). Plasma samples were simply pretreated with methanol for deproteinization. Chromatographic separation was performed on an Agilent Zorbax C₁₈ column with a mobile phase of 10 mM ammonium acetate buffer containing 0.1% formic acid–methanol (20:80, v/v) at a flow rate of 1 mL min⁻¹. The electrospray ionization (ESI) interface was employed in a single quadrupole mass spectrometer. The analytes were protonated in the positive ESI interface and detected in single ion monitoring (SIM) mode. Chromatographic separation was achieved in less than 3.5 min. The linearity was established over the range of 0.5–600 ng mL⁻¹. The lower limited of quantification (LLOQ) of the method was 0.5 ng mL⁻¹. The intra- and inter-run standard deviations were both less than 11.2%. The method was applied to study the pharmacokinetics of the mizolastine sustained-release tablets in healthy volunteers.     

IC Determination of Halide Impurities in Ionic Liquids

An ion chromatographic (IC) method has been developed for determination of trace levels of halide impurities in various types of ionic liquids (ILs). The advantage of this method is that all relevant halide species can be measured in a single chromatographic analysis. Separation of halides was performed on a Dionex AS9-HC column using an eluent consisting of 20 mM NaOH and 10% (v/v) acetonitrile, delivered at 1.5 mL min⁻¹. Using this eluent, fluoride, chloride and bromide were well resolved from each other, but iodide was co-eluted with tetrafluoroborate (BF₄⁻) present as a counter-anion in tetrafluoroborate-based ILs. The same eluent was also used successfully for the determination of halides in highly hydrophobic ILs, such as those based on bis-(trifluoromethanesulfonyl)imide (TFSI⁻) and bis-perfluoroethylsulfonylimide (BETI⁻). In this case, 50% (v/v) acetonitrile aqueous was needed to dissolve the sample before injection, and this did not adversely affect the separation. Detection limits in the measured solution were 0.1, 0.2 and 1.0 ppm for chloride, bromide and iodide, respectively, by conductivity detection, and 0.02 ppm for iodide by UV detection.     

Quantitative Determination of Acyclovir in Aqueous Humor by LC-MS

A specific, sensitive and precise liquid chromatographic assay method was established using LC-MS for the determination of acyclovir (ACV) in aqueous humor (AH), which was directly injected onto an Inertsil ODS-3 C₁₈ column without any pretreatment. The Agilent 1100 series LC–MS system was operated under the electrospray ionization mode (ESI). The analyte was separated from endogenous substances with a mobile phase of methanol: water: acetic acid (5:95:0.1, v/v) at a flow-rate of 0.3mL min⁻¹. A linear response was observed over the concentration range from 5 to 50ng mL⁻¹ (r=0.9993). Intra- and inter-day coefficients of variation were in the ranges 5.2–9.0% and 5.8–8.2%, respectively. The recovery was > 91.0%, and the limit of detection was approximate 1ng mL⁻¹. The pharmacokinetics of topically applied eye-drop and thermosetting gel were compared in rabbits utilizing the present method, the results demonstrated that LC-MS was a powerful tool for the detection of ACV in AH.     

HPLC Determination of DCJW in Rat Plasma and Its Application to Pharmacokinetics Studies

A high-performance liquid chromatography–UV method for determining DCJW concentration in rat plasma was developed. The method described was applied to a pharmacokinetics study of intramuscular injection in rats. The plasma samples were deproteinized with acetonitrile in a one-step extraction. The HPLC assay was carried out using a VP-ODS column and the mobile phase consisting of acetonitrile–water (80:20, v/v) was used at a flow rate of 1.0 mL min⁻¹ for the effective eluting DCJW. The detection of the analyte peak area was achieved by setting a UV detector at 314 nm with no interfering plasma peak. The method was fully validated with the following validation parameters: linearity range 0.06–10 μg mL⁻¹ (r > 0.999); absolute recoveries of DCJW were 97.44–103.46% from rat plasma; limit of quantification, 0.06 μg mL⁻¹ and limit of detection, 0.02 μg mL⁻¹. The method was further used to determine the concentration–time profiles of DCJW in the rat plasma following intramuscular injection of DCJW solution at a dose of 1.2 mg kg⁻¹. Maximum plasma concentration (C _max) and area under the plasma concentration–time curve (AUC) for DCJW were 140.20 ng mL⁻¹ and 2405.28 ng h mL⁻¹.     

Simultaneous Determination of Olanzapine and Fluoxetine by HPLC

A rapid, specific reversed phase HPLC method has been developed for simultaneous determination of olanzapine and fluoxetine in their formulations. Chromatographic separation of these two pharmaceuticals was carried out on an Inertsil C₁₈ reversed phase column (150 mm × 4.6 mm, 5 μm) with a 40:30:30 (v/v/v) mixture of 9.5 mM sodium dihydrogen phosphate (pH adjusted to 6.8 ± 0.1 with triethylamine), acetonitrile and methanol as mobile phase. The flow rate 1.2 mL min⁻¹ and the analytes are monitored at 225 nm. Paroxetine was used as internal standard. The assay results were linear from 25 to 75 μg mL⁻¹ for olanzapine (r ² ≥ 0.995) and 100–300 μg mL⁻¹ for fluoxetine (r ² ≥ 0.995), showed intra- and inter-day precision less than 1.0%, and accuracy of 97.7–99.1% and 97.9–99.0%. LOQ was 0.005 and 0.001 μg mL⁻¹ for olanzapine and fluoxetine, respectively. Separation was complete in less than 10 min. Validation of the method showed it to be robust, precise, accurate and linear over the range of analysis.     

High-Performance Liquid Chromatographic Determination of Cefepime and Cefazolin in Human Plasma and Dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of cefepime and cefazolin in human plasma and dialysate. For component separation, the method utilized a C₁₈ column with an aqueous mobile phase of dibasic potassium hydrogen phosphate (pH 7.0) and methanol gradient at a flow rate of 1 mL min⁻¹. The method demonstrated linearity from 2.0 to 100.0 μg mL⁻¹ (r > 0.999) with detection limit of 1 μg mL⁻¹ for both cefepime and cefazolin. The method was utilized for evaluation of plasma and dialysate samples in a clinical study evaluating the dialyzer clearance of cefepime and cefazolin using high-flux hemodialysis with varying blood flow rates in chronic kidney failure patients undergoing hemodialysis and peritoneal dialysis treatment.     

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometric detection (SPE–CE–MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE–CE–MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L⁻¹ ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 μL min⁻¹ was isopropanol–water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08–10 ng mL⁻¹. Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL⁻¹. Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.     

Analysis of Terbumeton and its Major Metabolites by SPE and DAD-HPLC in Soil Bulk Water

A rapid and inexpensive method for simultaneous quantification of terbumeton (TER), and its major potential metabolites (TED; terbumeton-desethyl, TOH; terbumeton-2-hydroxy and TID; terbumeton-deisopropyl) in soil bulk water (SBW) samples is proposed. The analytical method involves extraction–concentration from SBW samples using a graphitized carbon black (GCB) cartridge followed by their separation–detection by reversed-phase high-performance liquid chromatography analysis using a C₁₈ column and a diode array detector. A mobile phase of acetonitrile−0.005 mol L⁻¹ phosphate buffer (pH 7.0) (35:65, v/v) at a flow rate of 0.8 mL min⁻¹ in isocratic elution mode has been used. After optimization of the extraction and separation conditions, this method can be used for the simultaneous determination of investigated compounds in the range of the international limits of 0.1 μg L⁻¹. For TER the detection limit was 0.009 μg L⁻¹ and it was 0.100, 0.550, and 0.480 μg L⁻¹ for TED, TOH, and TID, respectively. The recoveries of TER, TED, TOH, and TID from SBW samples, measured at three levels of concentration range, were found to be between 48.0 and 102.0%. The intra-day precision measured by relative standard deviation (RSD) was always lower than 9.0%.     

Isolation and Simultaneous Determination of Coumarin Compounds in Radix Angelica dahurica

For the first time simple, rapid, and systematic methods have been established for preparative isolation and purification of coumarin compounds in an important traditional Chinese Medicine, Radix Angelica dahurica, and for simultaneous determination of several of the compounds in the medicine. Bergapten, imperatorin, and cnidilin, three of the biologically active coumarin compounds, were isolated from the chloroform-soluble fraction of the ethanol extract of Radix Angelica dahurica. After further purification by open column ODS chromatography the purified components were simultaneously determined, with two other coumarins (osthole and isoimperatorin), by reversed phase high-performance liquid chromatography (RP-HPLC) on a C₁₈ column, with methanol–water, 66:34 (v/v), as mobile phase at a flow rate of 0.8 mL min⁻¹. The compounds were detected by UV absorption at 310 nm. Calibration plots for all the coumarins had correlation coefficients close to unity. Limits of detection (S/N = 3) were <92 ng mL⁻¹ and limits of quantification (S/N = 10) were <259 ng mL⁻¹. Mean recovery of the coumarins was in the range 96.7–101.9% and the intra-day and inter-day precision, as relative standard deviation, was <2.3 and <2.9%, respectively. This simple, sensitive, and reproducible method can be used for quality control of Radix Angelica dahurica.     

Development of a Validated Stability-Indicating LC Method for Nitazoxanide in Pharmaceutical Formulations

A reversed-phase liquid chromatographic (LC) method was developed for the assay of nitazoxanide (NTZ) in solid dosage formulations. An isocratic LC separation was performed on a Phenomenex Synergi Fusion C₁₈ column (250 mm × 4.6 mm, i.d., 4 μm particle size) using a mobile phase of 0.1% o-phosphoric acid solution, pH 6.0: acetonitrile (45:55, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was achieved with a photodiode array detector at 240 nm. The detector response for NTZ was linear over the concentration range from 2 to 100 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method were proved using stress conditions. The RSD values for intra-day precision were less than 1.0% for tablets and powder for oral suspension. The RSD values for inter-day precision were 0.6 and 0.7% for tablets and powder for oral suspension. The accuracy was 100.4% (RSD = 1.8%) for tablets and 100.9% (RSD = 0.3%) for powder for oral suspension. The limits of quantitation and detection were 0.4 and 0.1 μg mL⁻¹. There was no interference of the excipients on the determination of the active pharmaceutical ingredient. The proposed method was precise, accurate, specific, and sensitive. It can be applied to the quantitative determination of drug in tablets and powder for oral suspension.     

Modulation of mobile phase composition in flow-injection/sequential-injection chromatography exploiting multisyringe flow analysis

In this paper, a time-based multicommutated flow system is proposed for appropriate selection and modulation of mobile phase composition in flow-injection (FI)/sequential-injection (SI) chromatography. The novel flow assembly involves the on-line coupling of a short monolithic reversed-phase chromatographic column with a multisyringe flow injection set-up furnished with a set of solenoid valves. The proposed hyphenated technique was applied to the simultaneous spectrophotometric determination of thiamine (B₁), pyridoxine (B₆) and cyanocobalamin (B₁₂) which were taken as model analytes. The separation method capitalizes on a dual isocratic elution protocol involving the use of a single forward stroke of the multisyringe pump for initial delivery of 50 mmol L⁻¹ ammonium acetate (pH 7.0) for 2.4 min followed by 50 mmol L⁻¹ ammonium acetate–methanol (80:20, v/v) for 6.4 min at 0.5 mL min⁻¹ and room temperature. Detection was performed at the maximum wavelength for each target vitamin—280 nm for B₁, 325 nm for B₆, and 360 nm for B₁₂. A first-order, two-level full-factorial design was utilized to ascertain the significant variables influencing the chromatographic separation and the magnitude of the interaction effects. The experimental design method revealed that resolution of the target vitamins is highly dependent on the pH, percentage of organic modifier, and their second-order interaction. The multisyringe flow-injection-based monolithic column separation method, which should be viewed as an expeditious and cost-effective alternative to the high-performance liquid chromatography counterpart, was applied to the separation and determination of B₁, B₆, and B₁₂ in different pharmaceutical dosage forms in less than 9 min. Statistical comparison of the results from the proposed procedure with those from the HPLC method endorsed by the US Pharmacopeia revealed there were no significant differences at the 95 % confidence level.     

LC Method for Studies on the Stability of Sibutramine in Soft Gelatin Capsules

A sensitive and rapid liquid chromatographic method was successfully developed and validated for the determination of sibutramine hydrochloride in bulk and capsules. Sibutramine in the presence of its degradation products was analyzed using UV detection at 225 nm. Chromatography was performed on a reversed-phase C₈ (150 × 4.0 mm I.D., 5 μm) analytical column under isocratic conditions. The mobile phase was composed of acetonitrile:water (aqueous phase containing 0.3% triethylamine and pH adjusted to 7.0) (75:25, v/v) at a flow-rate of 1.1 mL min⁻¹. No chromatographic interference was found during the analysis. Light was the stress condition which most contributed to sibutramine degradation. The method showed a linear response (r > 0.999) from 30 to 90 μg mL⁻¹. The mean recovery for capsules was 101.2%. Inter-day assays showed relative standard deviations of 0.42 and 1.62% for bulk and capsules, respectively. The developed method is able to separate sibutramine from its major degradation products and it may be used in the quality control of this active pharmaceutical ingredient in both bulk and capsules.     

Liquid chromatographic determination of erythromycins in fermentation broth

Summary A simple and sensitive isocratic LC method is described for the determination of erythromycins in fermentation broths. A simple technique utilizing acetone-methyl ethyl ketone, 1∶1, as extraction solvent was coupled with suitable chromatographic conditions—compounds were separated on a 250 mm×4.6 mm i.d., 5 μm, reversed-phase column at 65°C with acetonitrile-0.2m K₂HPO₄ pH7.0-water, 35:5:60 (v/v), as mobile phase at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. Separation of erythromycin F from polar components of the fermentation liquid was sufficient. Erythromycins A, B, C, D, and E, andN-desmethylerythromycin A were also separated, as were known decomposition products of erythromycin A and several unknown components. The method is suitable for monitoring the progress of erythromycin fermentation.     

A Validated Chiral LC Method for the Enantiomeric Separation of Repaglinide on Amylose Based Stationary Phase

A simple, isocratic, normal phase chiral HPLC method was developed and validated for the enantiomeric separation of repaglinide, (S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid, an antidiabetic in bulk drug substance. The enantiomers of repaglinide were resolved on a ChiralPak AD-H (amylose based stationary phase) column using a mobile phase consisting of n-hexane: 2-propanol:trifluoroacetic acid (95:5:0.2 v/v/v) at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be not >3.5 in optimized method. The presence of trifluoroacetic acid in the mobile phase played an important role, in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The calibration curve for (R)-enantiomer showed excellent linearity over the concentration range of 900 ng mL⁻¹ (LOQ) to 6,000 ng mL⁻¹. The limit of detection and limit of quantification for (R)-enantiomer were 300 and 900 ng mL⁻¹, respectively. The percentage recovery of the (R)-enantiomer ranged between 98.3 and 101.05% in bulk drug samples of repaglinide. Repaglinide sample solution and mobile phase were found to be stable up to 48 h. The developed method was found to be enantioselective, accurate, precise and suitable for quantitative determination of (R)-enantiomer in bulk drug substance.