Development and validation of column high-performance liquid chromatographic and ultraviolet spectrophotometric methods for citalopram in tablets

Column high-performance liquid chromatographic (LC) and UV spectrophotometric methods for the quantitative determination of citalopram, a potent and selective serotonin reuptake inhibitor, in tablets were developed. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by the reversed-phase technique on an ACE C18 column with a mobile phase composed of 0.30% triethylamine solution-acetonitrile (55 + 45, v/v) adjusted to pH 6.6 with 10% ortho-phosphoric acid at a flow rate of 1.0 mL/min and 25 degrees C. The UV spectrophotometric method was performed at 239 nm. The linearity of the LC method was in the range of 10.00-70.00 microg/mL, and 2.50-17.50 microg/mL for the UV spectrophotometric method. The interday and intraday assay precision was < 1.5% (relative standard deviation) for the LC and UV spectrophotometric methods. The recoveries were in the range 100.70-101.35% for the LC method and 98.48-98.65% for the UV spectrophotometric method. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantitation of citalopram in tablets.     

Biological assay and liquid chromatographic method for analysis of moxifloxacin in tablets

A microbiological assay and a liquid chromatographic method were validated for quantitation of moxifloxacin in tablets. The microbiological method consisted of a cylinder-plate agar diffusion assay using Micrococcus luteus ATCC 9341 as the test microorganism and phosphate buffer (0.1M, pH 8.0) as the diluent solution. The response graphs for standard and sample solutions were linear (r = 0.9479), and no parallelism deviations were detected in the tested levels of concentration (4.0, 8.0, and 16.0 microg/mL). The interday precision was 2.73%. Recovery values were between 96.25 and 100.5%. The chromatographic analyses were performed using a Shim-pack CLC-ODS column (250 x 4.6 mm, 5 microm) with a mobile phase consisting of (A) a mixture of phosphoric acid (0.17%, v/v) with tetramethylammonium hydroxide (0.05M) and acetonitrile (95 + 5, v/v) and (B) methanol (55 + 45, v/v) adjusted to pH 3.0. The flow rate was 1.0 mL/min, and detection was made at 294 nm. The method was linear in a range from 12.0 to 42 microg/mL (r = 0.9999), and the interday precision was 1.39%. Recovery ranged between 101.9 and 103.81%. Both validated methods were used to quantify the moxifloxacin content in tablets exposed to ultraviolet radiation, and similar results were obtained.     

Stereochemical analysis of betamethasone and dexamethasone by derivatization and high-performance liquid chromatography.

A simple and economical high-performance liquid chromatographic method has been developed for the simultaneous determination of betamethasone and dexamethasone. The method is based on the derivatization of the structural epimers of betamethasone and dexamethasone with a homochiral reagent, N-carbobenzoxy-L-phenylalanine. The derivatives obtained were easily recognized by a non-chiral silica column with n-hexane-dichloromethane-isopropanol (100:100:4, v/v/v) as a mobile phase and a good separation was obtained for quantitation. The method was satisfactorily applied to the determination of betamethasone and dexamethasone in tablets.     

Sensitive determination of MDMA and its metabolite MDA in rat blood and brain microdialysates by HPLC with fluorescence detection

Simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high-performance liquid chromatography with fluorescence detection (HPLC-FL) was developed. Microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The DIB-derivatives of MDMA, MDA and the internal standard, 1-methyl-3-phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)-acetonitrile-methanol-2-propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5-500 and 5.0-1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra- and the inter-assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated.     

Quantitation of glucosamine from shrimp waste using HPLC

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.     

Development and validation of a high performance liquid chromatographic method for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets

A simple, sensitive and rapid high performance liquid chromatographic method was developed and validated for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets. Chromatographic separation and detection was carried out on a C-18 column using 0.05 M potassium dihydrogen phosphate buffer (pH 5.0) and acetonitrile in the ratio of 94: 06 (v/v) as mobile phase at wavelength of 225 nm. The method was linear in the concentration range of 3.75–22.5 μg/mL for potassium clavulanate and 15–90 μg/mL for cefadroxil. The flow rate was 1.0 mL/min and the total analysis time was less than 10 min. The mean recoveries was found to be greater than 99% with RSD less than 1.0%. The proposed method was validated by performing linearity, recovery, specificity, robustness, LOD/LOQ and within-day and between-day precision. The chromatographic results obtained from the synthetically prepared tablets show that the method is highly precise and accurate for the simultaneous quantitation of clavulanate potassium and cefadroxil.     

Thermostability testing and degradation profiles of doxycycline in bulk, tablets, and capsules by HPLC

A high-performance liquid chromatography (HPLC) method for the quantitation of doxycycline in bulk, tablets, and capsules after storage at -20, 5, 25, 40, 50, 60, and 70 degrees C, has been developed and validated. The samples are eluted from a micro-Bondapak C8 column (4.6x150-mm, i.d., 5-microm particle size) at 27 degrees C, with a mobile phase of acetonitrile-water-THF (29.5:70:0.5, v/v/v), adjusted to pH 2.5 with 1.0M HCl. The flow rate is 1.0 mL/min and detection by UV is at 350 nm. The stability of doxycycline in bulk and in pharmaceuticals is checked over 90 days. Doxycycline shows thermo-degradation after exposure to high temperature; tablets are more stable than capsules. The shelf lives (t90%) are determined to be 1.00, 2.84, and 5.26 years in bulk, capsules, and tablets, respectively, at 25 degrees C. Metacycline and 6-epidoxycyline are identified as degradation products at high temperatures. Amounts of doxycycline, metacycline, and 6-epidoxycycline in all samples are determined by HPLC, and the results compare with those from micellar electrokinetic capillary chromatography. After 90 days, metacycline and 6-epidoxycyline are almost equal in test samples from standard bulk form, tablets, and capsules. It is 27.8+/-0.3%, 13.7+/-0.1%, and 18.8+/-0.2%, respectively.     

A validated enantioselective HPLC assay of dexibuprofen in dexibuprofen tablet formulations

A high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed and validated for the quantitation of dexibuprofen in dexibuprofen tablets using ovomucoid chiral stationary phase (Ultron ES-OVM). The mobile phasewas composed of 0.025 M potassium phosphate dibasic (pH 4.5)-methanol-ethanol (85:10:5 v/v/v). The method was validated for specificity, linearity, range, accuracy, precision and robustness. The method was enantiomerspecific for the determination of dexibuprofen [S-(+)-isomer ibuprofen] in the presence of R-(-)-isomer ibuprofen in bulk drug, pharmaceutical dosage form and under stress degradation. The method was linear over the range 15-35 mg/mL with r2 = 0.9995; accuracy and precision were acceptable with %RSD < 2.0%. The method was found to be specific, precise, accurate, robust and stability-indicating, and can be successfully applied for the routine analysis of dexibuprofen in bulk drug and pharmaceutical dosage form.     

Chromatographic Application on Calixarene Bonded Stationary Phases: A Stability Indicating LC-Method for Determination of Celecoxib in Tablet Formulation

A method is described for extraction and quantification of celecoxib in tablets. The extraction was achieved through centrifugation of the fine powder of the tablets in Acetonitrile (ACN). The extract was examined by LC. The chromatographic separation was carried out on a Caltrex AIII column, a relatively new packing material consisting of silica-bonded calix[8]arene, using isocratic binary mobile phase of ACN and H₂O (55%:45%, v/v). A diode array detector was used at 254 nm for detection. The method was validated for system suitability, linearity, precision, limits of detection and quantitation, specificity, stability and robustness. The limits of detection and quantitation were 0.122 and 0.488 μg mL^?1, respectively. The recovery value of this method was 101.88% and the reproducibility was within 2.08.     

Chemical profile of meta-chlorophenylpiperazine (m-CPP) in ecstasy tablets by easy ambient sonic-spray ionization, X-ray fluorescence, ion mobility mass spectrometry and NMR

Meta-chlorophenylpiperazine (m-CPP) is a new illicit drug that has been sold as ecstasy tablets. Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) and X-ray fluorescence spectrometry (XRF) are shown to provide relatively simple and selective screening tools to distinguish m-CPP tablets from tablets containing amphetamines (mainly 3,4-methylenedioxymethamphetamine (MDMA)). EASI-MS detects the active ingredients in their protonated forms: [m-CPP + H](+) of m/z 197, [MDMA + H](+) of m/z 194, and [2MDMA + HCl + H](+) of m/z 423 and other ions from excipients directly on the tablet surface, providing distinct chemical fingerprints. XRF identifies Cl, K, Ca, Fe, and Cu as inorganic ingredients present in the m-CPP tablets. In contrast, higher Cl concentrations and a more diverse set of elements (P, Cl, Ca, Fe, Cu, Zn, Pt, V, Hf, Ti, Pt, and Zr) were found in MDMA tablets. Principal component analysis applied to XRF data arranged samples in three groups: m-CPP tablets (four samples), MDMA tablets (twenty three samples), and tablets with no active ingredients (three samples). The EASI-MS and XRF techniques were also evaluated to quantify m-CPP in ecstasy tablets, with concentrations ranging from 4 to 40 mg of m-CPP per tablets. The m-CPP could only be differentiated from its isomers (o-CPP and for the three isomers p-CPP) by traveling wave ion mobility mass spectrometry and NMR measurements.     

Simultaneous determination of gliquidone, pioglitazone hydrochloride, and verapamil in formulation and human serum by RP-HPLC

In the present study, a reverse-phase high performance liquid chromatography method was developed, validated and applied for the simultaneous determination of gliquidone, pioglitazone hydrochloride and verapamil in tablets and human serum. Chromatographic separation was achieved on a C18 column (5 μm, 25 × 0.46 cm) with a mobile phase consisting of methanol-water-acetonitrile (80:10:10 v/v/v) with a flow rate of 0.7 mL/min and pH adjusted to 3.50 with phosphoric acid at 230 nm. Glibenclamide was used as internal standard. The experimentally derived limit of detection and limit of quantitation were determined to be 0.24, 0.93, 0.40, and 0.80, 3.11, 1.36 μg/mL for gliquidone, pioglitazone, and verapamil, respectively. There were no interfering peaks due to the excipients present in the pharmaceutical tablets. Thus, the proposed method is simple and suitable for the simultaneous analysis of active ingredients in dosage forms and human serum.     

Analysis of ketorolac and its related impurities by capillary electrochromatography

Capillary electrochromatography (CEC) was employed for the assay of ketorolac (KT) and its known related impurities [1-hydroxy analog of ketorolac (HK), 1-keto analog of ketorolac (KK), ketorolac decarboxylated (DK)] in both drug substance and coated tablets. Detection was made at 323 nm and flufenamic acid was selected as internal standard. The experiments were performed in a 100 microm i.d. capillary packed with RP-18 silica particles (33.0, 24.5, 23.0 cm total, effective and packed lengths, respectively). The composition of the mobile phase was optimised by changing pH of the buffer and acetonitrile (ACN) content and by addition of other organic modifiers (methanol, ethanol, isopropanol, n-propanol) in order to evaluate the effect of these factors on the method performance (efficiency, retention and resolution). The optimum mobile phase consisted of a mixture of 50 mM ammonium formate buffer pH 3.5-water-acetonitrile (10:20:70, v/v/v), while voltage and temperature were set at 30 kV and 20 degrees C, respectively. Applying these conditions, all peaks were baseline resolved and the analysis was performed in less than 9 min. Selectivity, repeatability of retention time and peak area, detection and quantitation limits, linearity and range, precision and accuracy were also investigated. R.S.D. and bias values obtained for all the analytes were below 5% and sensitivity was satisfactory, thus the method was deemed suitable for pharmaceutical quality control. Applying the method to coated tablets, a recovery of 98.5+/-0.8% and an R.S.D. of 0.5% were found.     

Determination of 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine enantiomers in whole blood

A method for the determination of the enantiomeric content of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in microsamples (200 microliters) of whole blood is described. The method involves liquid-liquid extraction of MDA and MDMA from blood and derivatization with the chiral reagent N-trifluoroacetyl-L-prolyl chloride. Separation, identification and quantitation of diastereomeric derivatives is by gas chromatography-mass spectrometry. The analytical range of the assay is from 0.12 ng to 48 ng injected on-column. Details for the synthesis of the enantiomers of MDMA are also provided.     

Simultaneous determination of metformin in combination with rosiglitazone by reversed-phase liquid chromatography

A simple, rapid, and precise reversed-phase liquid chromatographic method is developed for the simultaneous determination of metformin in combination with rosiglitazone. This method uses a Zorbax XDB C(18) 15-cm analytical column, a mobile phase of acetonitrile and buffer containing 10mM disodium hydrogen phsosphate, and 5mM sodium dodecyl sulphate in the ratio of 34:66 (v/v), and pH is adjusted to 7.1 with orthophosphoric acid. The instrumental settings are a flow rate of 1 mL/min, column temperature at 40 degrees C, and detector wavelength of 226 nm. The internal standard method is used for the quantitation of metformin and rosiglitazone. Methylparaben is used as an internal standard. The method is validated and shown to be linear. The correlation coefficients for metformin and rosiglitazone are 0.9996 and 0.9997, respectively. The relative standard deviation for six replicate measurements in two sets of each drug in the tablets is always less than 2%.     

Stability evaluation of tramadol enantiomers using a chiral stability-indicating capillary electrophoresis method and its application to pharmaceutical analysis

In this study, a chiral stability-indicating CE assay was developed for the stability evaluation of tramadol (TR) enantiomers in commercial tablets using maltodextrin as chiral selector. To investigate the stability-indicating power of the analytical method as well as stability evaluation of TR enantiomers, active pharmaceutical ingredient and TR tablets were subjected to photolysis, heat, oxidation and hydrolysis to conduct stress testing. Best separation for the TR enantiomers was achieved on an uncoated fused-silica capillary at 20 °C using borate buffer (50 mM, pH 10.2) containing 10% m/v maltodextrin. All determinations were performed by a UV detector at 214 nm. A constant voltage of 20 kV was applied to obtain the separation. The range of quantitation for both enantiomers was 5-100 μg/mL (R>0.996). Intra- and inter-day RSD (n=6) were less than 10%. The percent relevant errors were obtained to be less than 4.0 for both enantiomers. The limits of quantitation and detection for both enantiomers were 5 and 1.5 μg/mL, respectively. Degradation products resulting from the stress studies were the same for both enantiomers and did not interfere with the detection of the enantiomers.     

A selective high-performance liquid chromatography method for the determination of reboxetine in bulk drug and tablets

Reboxetine is used as a selective noradrenaline reuptake inhibitor for the treatment of major depressive disorders. It is effective in the treatment of severe depression and safer to use than traditional tricyclic antidepressants. In this study, a novel, simple, and rapid stability-indicating high-performance liquid chromatography (HPLC) method for reboxetine methansulfonate was successfully developed and validated for the assay of tablets. The method was used to quantify reboxetine in tablets; it employed a C18 column (150 x 4.6 mm id) with an isocratic mobile phase consisting of methanol-phosphate buffer (pH 7, 0.02 M; 55 + 45, v/v) at a flow rate of 1.0 mL/min. Reboxetine was detected by an ultraviolet detector at 277 nm. The retention time of reboxetine was about 4.5 min. The developed HPLC method was validated with respect to linearity, precision, sensitivity, accuracy, and selectivity. The method was linear over the concentration range 1-50 microg/mL (r = 0.9999). The limits of detection and the quantitation of reboxetine were 0.1 and 0.3 microg/mL, respectively. The relative standard deviation values for intraday and interday precision were 0.78-1.01 and 1.08-1.37%, respectively. Selectivity was validated by subjecting a stock solution of reboxetine to neutral, acid, and alkali hydrolysis, as well as oxidation, dry heat treatment, and photodegradation. The peaks of the degradation products did not interfere with the peak of reboxetine. The results indicated that the proposed method could be used in a stability assay. The proposed method was successfully applied to the determination of reboxetine in tablets. Excipients present in the tablets did not interfere with the analysis.     

High-performance liquid chromatography with fluorescence detection for the simultaneous determination of 3,4-methylenedioxymethamphetamine, methamphetamine and their metabolites in human hair using DIB-Cl as a label

This paper describes a highly sensitive HPLC method for the simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in human hair samples. The amphetamines investigated were derivatized with the fluorescent reagent, DIB-Cl to yield highly fluorescent DIB-derivatives, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 nm and 430 nm, respectively. The separation was achieved on an ODS column with an isocratic mobile phase composed of acetonitrile-methanol-water (30:40:30, v/v/v). The limits of detection for the four compounds obtained by the proposed method ranged from 11 to 200 pg/mg. The method was successfully applied to the determination of MDMA and MDA in hair samples obtained from MDMA abuser.     

Quantification of MDMA and MDA in abusers' hair samples by semi-micro column HPLC with fluorescence detection

A sensitive semi-micro column high-performance liquid chromatography with fluorescence detection method was developed for the determination of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MP) and amphetamine (AP) in human hair. 4-(4,5-Diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and 1-methyl-3-phenylpropylamine were used as labeling reagent and internal standard, respectively. These drugs were extracted from hair into 5% trifluoroacetic acid in methanol, and fluorescent labeled with DIB-Cl. The separation of DIB-derivatives was achieved on a reversed-phase semi-micro ODS column with an acetonitrile-methanol-water (30:40:30, v/v/v%) mixture as a mobile phase. The limits of detection at a signal-to-noise ratio of 3 for MDMA, MDA, MP and AP were 0.25, 0.15, 0.25 and 0.19 ng/mg, respectively. Precision of intra- and inter-day assay as the relative standard deviation were in the range 1.5-6.8% (n = 5) and 2.7-4.7% (n = 5), respectively. The proposed method was highly sensitive and able to detect MDMA and its related compounds in small amounts of hair sample, and could be applied to quantification of six abusers' hair samples.     

Simultaneous high-performance liquid chromatographic determination of pioglitazone and metformin in pharmaceutical-dosage form

A simple, rapid, and precise method is developed for the quantitative simultaneous determination of metformin and pioglitazone in a combined pharmaceutical-dosage form. Separation is achieved with a Zorbax XDB C(18), 15-cm analytical column using buffer-acetonitrile (66:34, v/v) of pH 7.1, adjusted with orthophosphoric acid as the mobile phase. The buffer used in the mobile phase contains 10mM disodium hydrogen phosphate and 5mM sodium dodecyl sulphate in double-distilled water. The instrumental settings are flow rate of 1 mL/min, column temperature at 40 degrees C, and detector wavelength of 226 nm. The internal standard method is used for the quantitation of the ingredients of this combination. Methylparaben is used as an internal standard. The method is validated and shown to be linear for metformin and pioglitazone. The correlation coefficients for metformin and pioglitazone are 0.9991 and 0.9999, respectively. The relative standard deviations for six replicate measurements in two sets of each drug in the tablets are always less than 2%.     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.