Conventional and micellar liquid chromatography method development for danazol and validation in capsules

Two isocratic liquid chromatographic methods (conventional and micellar) for the determination of danazol (DZ) in capsules using canrenone (CAN) as internal standard have been developed and validated. In conventional liquid chromatography a mobile phase 35% water:acetonitrile 65%, v:v, a flow-rate 1 ml min(-1) and a C18 Hypersil ODS (250 x 4.6 mm, 5 microm) column (25 degrees C) were used. In micellar liquid chromatography (MLC) the conditions were: mobile phase 40 mM sodium dodecyl sulfate:2% pentanol, flow-rate 0.5 ml min(-1) and C18 Hypersil ODS (150 x 3.0 mm, 5 microm) column (60 degrees C). For both methods. UV absorbance detection at 280 nm was used and a separation up to base line was achieved. Prior to HPLC analysis a simple sample preparation was required. The recoveries found in the accuracy test were 99 +/- 10 and 101 +/- 8%, in conventional liquid chromatography (CLC) and MLC, respectively. Repeatability and intermediate precision expressed as R.S.D. were lower than 5% for both methods. Detection limits obtained were 2.4 and 3.0 ng g(-1) in CLC and CLM, respectively.     

Separation and determination of Vitamins E and A in multivitamin syrup using micellar liquid chromatography and simplex optimization

A rapid method has been described for separation and determination of Vitamins A and E using micellar liquid chromatography (MLC). Influence of temperature of column and addition of organic modifiers on separation efficiency was investigated. A temperature of 30 degrees C and 1-butanol modifier was selected. Optimization of the parameters affecting the separation including percent of organic modifier, pH of the mobile phase, concentration of surfactant, and flow rate of the mobile phase was performed simultaneously using the super-modified simplex (SMS) procedure. Results showed that 11.7% 1-butanol, 76.9 mM sodium dodecyl sulphate (SDS), pH of 6.73 and a flow rate of 1.35 ml min(-1) are the best conditions for separation of these compounds. The analytical parameters including linearity, r>0.9990; limit of detection 1.71 and 4.52 microg ml(-1) for A and E, respectively; precision of the method, R.S.D.<2.85%; and recovery, more than 90%, show that the method is useful for measuring these compounds in pharmaceutical preparations.     

Simultaneous high-performance liquid chromatographic determination of puerarin, daidzin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde and glycyrrhizin in Kampo medicines

We report a high-performance liquid chromatographic method to determine the quantities of puerarin, daidzin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde and glycyrrhizin in Kampo medicine. All seven compounds were separated in less than 30 min with a Wakosil-II 5C18 AR column by linear gradient elution using 0.01% (v/v) phosphoric acid acetonitrile (0 min 90:10, 10 min 88:12, 22 min 70:30, 30 min 30:70) as the mobile phase at a flow-rate of 1.0 ml/min(-1), and detection at 250 nm. The detection limits of these compounds are 0.15-0.3 microM with response linearity. This method was applied to determine the quantities in eight Kampo decoctions; Mao-to, Makyo-yokukan-to, Makyo-kanseki-to, Yokuinin-to, Sho-seiryu-to, Keima-kakuhan-to, Kakkon-to and Kakkon-to-ka-senkyu-sin'i. Glycyrrhizin content was lower in both the decoction and the methanol-diluted decoction of Sho-seiryu-to compared with the others. Low pH due to organic acids of Schisandrae fructus in the decoction caused inhibition for glycyrrhizin dissolution in Sho-seiryu-to.     

Optimization and validation of conventional and micellar LC methods for the analysis of methyltestosterone in sugar-coated pills

Two isocratic liquid chromatographic methods (conventional and micellar) for the determination of methyltestosterone in sugar-coated pills using fluoxymesterone as internal standard have been developed and validated. In conventional liquid chromatography a mobile phase 45% water:acetonitrile 55% (v:v), a flow-rate 1 mlmin(-1) and a C(18) Hypersil ODS (250 x 4.6 mm, 5 microm) column (25 degrees C) were used. In micellar liquid chromatography the conditions were: mobile phase 40 mM sodium dodecyl sulfate: 10% propanol, flow-rate 0.5 mlmin(-1) and C(18) Hypersil ODS (150 x 3.0 mm, 5 microm) column (60 degrees C). For both methods, UV absorbance detection at 245 nm was used and a separation up to base line was achieved. Prior to HPLC analysis a simple sample preparation was required.     

Micellar liquid chromatographic determination of five antianginals in pharmaceuticals

A procedure was developed for the determination of five antianginals (diltiazem, nadolol, nifedipine, propranolol and verapamil), using hybrid micellar mobile phases of sodium dodecyl sulphate (SDS) and pentanol, a C18 column and UV detection. All possible combinations of antianginals were resolved and determined using a mobile phase of 0.05 M SDS-5% pentanol with an analysis time of 9 min. Repeatabilities and intermediate precision were evaluated at four different drug concentrations in the 2-20 microg/ml (n=5) range. Limits of detection were in the range 0.028 microg/ml for diltiazem and 0.130 microg/ml for verapamil. The range of the limit of quantitation was from 0.092 to 0.431 microg/ml for the same compounds. Antianginal drugs were studied in pharmaceuticals with no interference from related compounds. The results of the analyses of pharmaceuticals formulations were in agreement with the declared compositions.     

Development and validation of a stability-indicating RP-HPLC method to separate low levels of dexamethasone and other related compounds from betamethasone

Betamethasone (BM) is an active pharmaceutical ingredient (API) or an intermediate which is used to manufacture various finished pharmaceutical products. Betamethasone is also used as a starting material to manufacture other APIs that are related to this steroid family. It is quite a challenging task to separate dexamethasone (DM) peak (the alpha epimer) and other structurally related compounds from BM. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate low levels of DM and other related compounds from BM and also from each other. This method was successfully validated for the purpose of conducting stability studies of betamethsone in quality control (QC) laboratories. The stability-indicating capability of this method was demonstrated by adequate separation of DM and all the degradation product peaks from BM peak and also from each other in aged stability samples of betamethasone. A gradient mobile phase system consisting of (A) water:acetonitrile (90:10, v/v) and (B) acetonitrile:isopropanol (80:20, v/v) was used with an ACE Phenyl column (10 cm × 4.6 mm, 3 μm particles, 100 Å pore size) and an ultraviolet (UV) detection at 240 nm.     

Optimised procedures for the reversed-phase liquid chromatographic analysis of formulations containing tricyclic antidepressants

The chromatographic behaviour (retention, selectivity, peak shape and resolution) of seven tricyclic antidepressants (TCAs), amitryptiline, clomipramine, doxepin, imipramine, maprotiline, nortryptiline and trimipramine, was examined. Conventional unendcapped Cs and C18 columns and an endcapped XTerra MS C18 column recommended for the analysis of basic compounds were used together with acetonitrile-water and micellar sodium dodecylsulfate (SDS)-pentanol mobile phases. The two best combinations were XTerra C18/acetonitrile, which yielded the largest efficiencies and resolution, and C8/SDS-pentanol, which eliminated the peak tails that were still observed with the XTerra C18 column. Both the systems were used to develop simple chromatographic procedures for the control of TCAs in pharmaceutical formulations using UV detection. The selected mobile phase compositions were 35% (v/v) acetonitrile (XTerra C18 column) and 0.075 M SDS-6% (v/v) pentanol (C8 column), both at pH 3. Satisfactory recoveries were achieved in both cases, with intra- and inter-day relative standard deviations (RSDs) always below 0.6 and 2.0%, respectively. The preparation of the samples was simple in both modes, since a previous extraction of the drugs was not needed. The micellar mode has, however, the advantage of using a smaller amount of organic solvent, which is retained in the micellar SDS solution. The C8 column is also less expensive.     

Analysis of pharmaceutical preparations containing antihistamine drugs by micellar liquid chromatography

Rapid chromatographic procedures for analytical quality control of pharmaceutical preparations containing antihistamine drugs, alone or together with other kind of compounds are proposed. The method uses C18 stationary phases and micellar mobile phases of cetyltrimethylammonium bromide (CTAB) with either 1-propanol or 1-butanol as organic modifier. The proposed procedures allow the determination of the antihistamines: brompheniramine, chlorcyclizine, chlorpheniramine, diphenhydramine, doxylamine, flunarizine, hydroxyzine, promethazine, terfenadine, tripelennamine and triprolidine, in addition to caffeine, dextromethorphan, guaifenesin, paracetamol and pyridoxine in different pharmaceutical presentations (tablets, capsules, suppositories, syrups and ointments). The methods require minimum handling sample and are rapid (between 3 and 12 min at 1 mLmin(-1) flow rate) and reproducible (R.S.D. values<5%). Limits of detection are lower than 1 microgmL(-1) and the recoveries of the analytes in the pharmaceutical preparations are in the range 100+/-10%.     

Separation and characterization of synthetic impurities of triclabendazole by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry

A simple high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) method for the separation and characterization of impurities in the synthesis of triclabendazole has been developed. The analytical separation was achieved on a reversed-phase C18 column using acetonitrile and water (60:40, v/v) as mobile solvent at a flow-rate of 1.0 ml/min at 25 degrees C, and an UV detection at 230 nm. The on-line HPLC/ESI-MSn examinations were performed using ion trap analyzer with extraction ion chromatography (EIC) technique in positive or negative ion modes. The semi-preparative separation was performed with a reversed-phase column using methanol and water (75:25, v/v) as mobile solvent at a flow-rate of 4 ml/min at 25 degrees C, and an UV detection at 230 nm. Thus, two impurities were detected and identified as 5-chloro-6-(2,3,4-trichlorophenoxy)-2-methylsulfanyl-1H-benzoimidazole and 5-chloro-6-(2,3-dichlorophenoxy)-1-methyl-2-methylsulfanyl-1H-benzoimidazole. Meanwhile, some intermediates of impurity-1 in multi-step synthetic reactions, were tracked. Structural elucidation by 1D and 2D NMR and ESI-MSn was discussed.     

Mathematical representation of analyte's capacity factor in binary solvent mobile phases using the Jouyban-Acree model

Applicability of a solution model, namely the Jouyban-Acree model, for mathematical representation of capacity factors of phenobarbital, phenytoin and carbamazepine in mobile phases containing water and the organic modifiers: methanol, acetonitrile, acetone and tetrahydrofuran and also a number of data sets collected from the literature has been shown. The accuracy of the proposed model is compared with those of the linear model and the quadratic equation using average percentage deviation (APD) as an accuracy criterion. The obtained mean and standard deviation of APDs of the Jouyban-Acree, linear and quadratic models are 8.1 +/- 8.4, 25.2 +/- 18.0 and 14.5 +/- 16.2%, respectively. The results showed that the Jouyban-Acree model provided more accurate calculations than the previously published models and the mean differences were statistically significant (p < 0.002).     

Development of an HPLC method for the determination of ranitidine and cimetidine in human plasma following SPE

A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for the determination of ranitidine and cimetidine in plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of investigated drugs were investigated. Sample preparation was carried out by adding an internal standard, famotidine, and the clean-up procedure was accomplished using solid-phase extraction (SPE). This method uses ultraviolet detection, the separation used a Lichrocart Lichrospher 60 RP-select B column and the mobile phase consisted of 0.2% triethylamine (TEA), 0.04 mol l(-1) KH2PO4 at pH 6.8 and 14% acetonitrile. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma. The method has been implemented to monitor ranitidine levels in clinical samples.     

Determination of 14 chemical constituents in the traditional Chinese medicinal preparation Huangqin-Tang by high performance liquid chromatography.

The high performance liquid chromatographic (HPLC) method for the identification and determination of baicalin (BG), wogonoside (WG), oroxylin-A-glucoside (OG), baicalein (B), wogonin (W), orxylin-A (O), paeoniflorin (PF), glycyrrhizic acid (GL), glycyrrhetinic acid (GA), liquiritin (LG), isoliquirition (ILG), liquiritigenin (L), isoliquiritigenin (IL) and ononin (ON) in Huangqin-Tang [Chinese characters: see text] was established. The samples were separated with a Wakosil C18 column (4.6 x 150 mm) by linear gradient elution using A (MeOH-HAC 100:1, v/v)-B (Water-HAC 100:1, v/v) (0 min, 30:70; 15 min, 40:60; 30 min, 60:40; 45 min, 80:20; 60 min, 100:0) as the mobile phase at a flow-rate of 1.0 ml/min. The detection was by diode-array UV/Vis detector (DAD), and the wavelength was set at the range of 200-400 nm. Satisfactory results were obtained within 60 min for the simultaneous determination of the 14 constituents. The repeatability (RSD) of the method was generally less than 2% (n=5, interday and intraday). The recovery of BG was 96.9+/-1.71, WG was 98.9+/-2.99, PF was 99.7+/-0.52, LG was 95.3+/-2.67, GL was 96.7+/-3.44, and GA was 94.8+/-4.16, respectively.     

Optimization of the separation of some Chelidonium maius L. alkaloids by reversed phase high-performance liquid chromatography using cyanopropyl bonded stationary phase

The high-performance liquid chromatographic method using two stationary phases: RP-18 and cyanopropyl silica for the analysis of some alkaloids in Chelidonium maius L. extracts is performed. The extract was chromatographed using a mobile phase composed of an organic modifier (acetonitrile, methanol, tetrahydrofuran or 1,4-dioxan), phosphate buffer and sodium octadecylsulfate or di (2-ethyl hexyl) orthophosphoric acid (HDEHP) as ion-pairing reagents. The influence of the different kind of stationary phase and organic modifiers on retention has been compared. In order to improve the selectivity of separation and peak shape, gradient elution was applied.     

Elevated serum tumor necrosis factor alpha and ferritin may contribute to the insulin resistance found in HCV positive Egyptian patients

OBJECTIVE: There is evidence of an increased incidence of insulin resistance and diabetes mellitus (DM) in patients with hepatitis C virus (HCV) infection. Several mechanisms have been proposed, including inadequate insulin secretion or interference with signaling within the insulin receptor. We assessed serum tumor necrosis factor alpha (TNFalpha) and ferritin levels as potential mediators of insulin resistance in HCV positive Egyptian patients. PATIENTS AND RESULTS: Patients (n = 27) with HCV infection, patients (n = 23) with hepatitis C and DM (HCV + DM), patients (n = 22) with DM, and sex- and age-matched controls (n = 18) were included in this study. The degree of insulin resistance (HOMA index) was significantly higher in the HCV, HCV + DM and DM groups compared to the controls. The mean +/- SD of the HOMA index was 4.53 +/- 2.84, 6.1 +/- 2.36, 3.69 +/- 2.2 and 1.32 +/- 0.49, in HCV, HCV + DM, DM and controls, respectively. Serum TNFalpha levels were significantly higher in the HCV, HCV + DM groups compared with the healthy controls and DM patients (p < 0.001). The median (range) values of TNFalpha in HCV, HCV + DM, DM patients and controls subjects were 25.5 (0.43-124.0), 19.8 (0.51-139), 0.85 (0-10.5) and 0.32 (0-5.8) pg/mL, respectively. There was a significant positive correlation between the HCV load and both HOMA index and TNF alpha level. HCV and HCV + DM patients also had significantly higher serum ferritin levels compared with healthy controls and patients with DM. The mean +/- SD of serum ferritin in HCV, HCV + DM, DM patients and controls subjects was 258.1 +/- 116.2, 285.8 +/- 124.3, 86.9 +/- 41.8 and 159.9 +/- 76.9 ng/mL, respectively. CONCLUSION: Patients with HCV infection had a significantly higher level of TNFalpha and ferritin which may explain their insulin resistance. HOMA index and serum TNFalpha levels correlated positively with the HCV load.     

A fast and efficient determination of amines and preservatives in cough and cold liquid and suspension formulations using a single isocratic ion-pairing high performance [correction of power] liquid chromatography method.

A single, highly selective ion-pairing reverse phase-high power liquid chromatography (RP-HPLC) method has been developed for the determination of amines and preservatives in a wide range of Tylenol((R)) liquid and suspension liquid products. As with many OTC products, the challenge is to quantitatively extract the analytes from difficult matrices and specifically analyze them in the presence of various excipients and flavors. Historically, separate analytical methods were used for each class of analytes (acids, bases and neutral compounds). In this method a mobile phase consisting of a buffered ion-pairing agent with acetonitrile, methanol and tetrahydrofuran was used to separate the charged amines from neutral and acidic compounds on a Phenomenex LUNA C8(2) 75 x 4.6 mm i.d. analytical column with a 3-microm particle size. The analytes include acids (benzoic acid), bases (pseudoephedrine, chlorpheniramine, dextromethorphan, doxylamine and diphenhydramine) and a neutral compound (butylparaben). The effects of pH, the chain length of the ion-pairing reagent, ionic strength and organic modifiers on the separation are discussed. The method is linear from 15 to 150% of the target amounts. The optimized method proves to be specific, robust and accurate for the analysis of the compounds.     

Simultaneous determination of baicalin, wogonoside, baicalein, wogonin, berberine, coptisine, palmatine, jateorrhizine and glycyrrhizin in Kampo medicines by ion-pair high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method for the simultaneous determination of four flavonoids, namely baicalin, wogonoside, baicalein and wogonin, and four berberine-type alkaloids, namely berberine, coptisine, palmatine and jateorrhizine, and glycyrrhizin in Kampo medicines is described. The analysis can be accomplished within 30 min with a Wakosil-II 5C18 HG column by linear gradient elution using a mobile phase containing aqueous phosphoric acid, sodium dodecyl sulfate and acetonitrile at a flow-rate of 1.0 ml x min(-1), a thermostatic oven at 45 degrees C, and detection at 265 nm. The method was applied to quantifying these components in three Kampo decoctions: Oren-gedoku-to, San'o-shashin-to and Hange-shashin-to. The decoctions were diluted with 65% methanol at the final stage because a large quantity of precipitate, mainly from baicalin and berberine, was formed. The within-day relative standard deviations were less than 2.02% (n=10). The recoveries of these compounds were 90.3-102%. The detection limits of these compounds were 0.02-1.96 microM per injection (5 microl).     

Determination of linsidomine in human plasma by tandem LC-MS with ESI

A sensitive method for the determination of linsidomine in plasma was developed, using high-performance liquid chromatographic (HPLC) separation with tandem mass spectrometric detection. Linsidomine was derivatised with propyl chloroformate and extracted with tert-butyl methyl ether/1,2-dichloroethane (55:45, v/v), back-extracted into HCl (0.01 M) followed by alkalinisation and back-extraction into ether; the final ether extract evaporated, reconstituted in mobile phase and then separated on a Phenomenex Luna C18 (2) 5 micron 2.1 x 150 mm column with a mobile phase consisting of methanol water formic acid (98/100%) (400:600:0.05, v/v/v) at a flow-rate of 0.4 ml min(-1). Detection was achieved by a Finnigan MAT mass spectrometer (LCQ) at unit resolution in the selected reaction monitoring (SRM) mode monitoring the transition of the protonated molecular ion m/z 257.0 to the product ion m/z 86.0. The mean recovery for linsidomine was 51% with a lower limit of quantification of 0.70 ng/ml using 1 ml plasma for extraction. This LC-MS/MS method for the determination of linsidomine in human plasma allows for better specificity and a higher sample throughput than the traditional LC-UV methods. It also demonstrates the profound effect that the composition of acidic modifiers and matrix constituents can have on the electrospray ionisation (ESI) of the analyte.     

高效液相色谱-质谱联用测定家兔体内可乐定血药浓度

目的用HPLC-MS测定家兔体内可乐定血药浓度,研究可乐定在家兔体内的药代动力学行为。方法 色谱仪为Waters Alliance 2790,色谱柱为XTerra C18(150 mm×2.1 mm ID,5 μm)。流动相为乙腈-碳酸氢铵水溶液,梯度洗脱;质谱仪为Micromass ZQ-4000仪,电喷雾电离源,选择离子监测(SIM)质荷比(m/z)为230。结果本方法回收率较高,重现性好。线性范围为1～80 μg·L^-1,最低检测浓度为0.05 μg·L^-1。主要药代动力学参数Cmax,AUC0-t和tmax分别为(27±9) μg·L^-1,(5 352±1 121) μg·L^-1,(79±17) h。结论本法灵敏度高,选择性强,具有较高的精密度和准确度。透皮贴剂中的可乐定可以平稳地透过家兔皮肤,稳定地控释达7 d。     

Determination of pioglitazone hydrochloride in bulk and pharmaceutical formulations by HPLC and MEKC methods

High Performance Liquid Chromatographic (HPLC) and Micellar Electrokinetic Chromatographic (MEKC) methods have been developed for the determination of pioglitazone, a new englycemic antidiabetic agent. Pioglitazone and its unsaturated impurity were separated by MEKC in less than 7 min using a 43 cm x 50 microm i.d. uncoated fused-silica capillary with extended light path for better sensitivity (25 kV at 30 degrees C) and a background electrolyte (BGE) consisting of 20% acetonitrile (v/v) in 20 mM sodium borate buffer pH 9.3 containing 50 mM sodium dodecyl sulphate (SDS). The influence of various parameters on the separation such as pH of the buffer, SDS concentration, buffer concentration, organic modifiers, temperature and voltage were investigated. The MEKC method was compared with HPLC method using a 5 microm symmetry C18 column (250 x 4.6 mm i.d.) eluted with a mobile phase consisting of a mixture of 50% (v/v) acetonitrile and 10 mM potassium dihydrogen phosphate buffer, adjusting the pH to 6.0 with 0.1 M KOH. The HPLC method is capable of detecting all process related compounds, which may be present at trace levels in finished products. Both methods were fully validated and a comparison was made. The results confirm that the methods are highly suitable for its intended purpose.     

Diastereomeric and enantiomeric high-performance liquid chromatographic separation of synthetic anisodamine

In order to investigate the enantiomeric pharmacokinetics and biotransformation of synthetic anisodamine (654-2), a cholinoceptor antagonist widely used in clinic in China, it has been preparatively separated into two racemates (I and II) by using ZORBAX Eclipse XDB-C18 column. The diastereo- and/or enantioseparations of 654-2, I and II were carried out by HPLC using CHIRALPAK AD-H as chiral stationary phase (CSP) and acetonitrile-2-propanol-DEA 97:3:0.1 (v/v/v) as mobile phase. The methods were optimized by studying mobile phase modifiers, concentration of modifier and column temperature. The HPLC method for the simultaneous separation of two pairs of enantiomers of 654-2 has been validated.