A new HPTLC densitometric method for analysis of swertiamarin in Enicostemma littorale and commercial formulations

A high-performance thin layer chromatographic densitometric method for the analysis of swertiamarin in 60% methanolic extract of Enicostemma littorale and commercial formulations has been developed and validated in this study. Swertiamarin was separated on aluminium-backed silica gel 60 F??? plates using ethyl acetate : methanol : water (77 : 15 : 8)% v/v as the mobile phase. This system was found to give a compact spot of swertiamarin at R(f) value 0.36 ± 0.01. The limit of detection and limit of quantification were found to be 31.25 and 103.12 ng spot?1, respectively. The proposed method was employed with a high degree of precision and accuracy for the estimation of swertiamarin in methanolic extract of Enicostemma littorale and in commercial formulations.     

HPTLC method for determination of nevirapine in pharmaceutical dosage form

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis of nevirapine both as a bulk drug and in formulations was developed and validated. The solvent system consisted of toluene-carbon tetrachloride-methanol-acetone-ammonia (3.5:3.5:2.0:1.0:0.05, v/v/v/v/v). Densitometric analysis of nevirapine was carried out in the absorbance mode at 289nm. This system was found to give compact spots for nevirapine (R(f) value of 0.44+/-0.02). Nevirapine was subjected to acid and alkali hydrolysis, oxidation, dry heat and wet heat treatment and photodegradation. The drug undergoes degradation under acidic, basic conditions and oxidation. Also the degraded products were well resolved from the pure drug with significantly different R(f) values. Linearity was found to be in the range of 30-1000ng/spot with significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots showed good linear relationship with r(2)=0.998+/-0.002 in the working concentration range of 300ng/spot to 1000ng/spot. The mean value of slope and intercept were 0.073+/-0.005 and 36.78+/-1.50, respectively. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 5 and 10ng/spot, respectively. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of acid degradation process. Arrhenius plot was constructed and activation energy was calculated.     

Determination of gatifloxacin and ornidazole in tablet dosage forms by high-performance thin-layer chromatography.

A simple and sensitive high-performance thin-layer chromatography (HPTLC) method has been developed for the quantitative estimation of gatifloxacin and ornidazole in its combined dosage forms. Gatifloxacin and ornidazole were chromatographed on silica Gel 60 F(254) TLC plate using n-butanol:methanol:ammonia (6 M) (8:1:1.5 v/v) as the mobile phase and scanned at 302 nm using a Camag TLC Scanner 3. The R(f) value of gatifloxacin and ornidazole was found to be 0.21 +/- 0.02 and 0.76 +/- 0.04, respectively. The linearity of gatifloxacin and ornidazole were in the range of 100 - 500 ng/spot and 250 - 1250 ng/spot, respectively. The limit of detection was found to be 40 ng/spot for gatifloxacin and 100 ng/spot for ornidazole. The proposed method was applied for the determination of gatifloxacin and ornidazole in combined dosage forms.     

Stability-indicating thin-layer chromatographic method for quantitative determination of ribavirin

A simple and accurate stability-indicating thin-layer chromatographic (TLC) method is developed and validated for the quantitative determination of ribavirin (RBV) in its bulk and with used for development consists of chloroform-methanol-acetic acid (60:15:15, v/v/v). The separated spots are visualized as bluish green spots after being sprayed with anisaldehyde reagent. RBV is subjected to different accelerated stress conditions. The drug is found to undergo degradation under all stress conditions, and the degradation products are well resolved from the pure drug with significantly different Rf values. The optical densities of the separated spots are found to be linear with the amount of RBV in the range of 5-40 microg/spot with a good correlation coefficient (r=0.9980). The limit of detection and limit of quantitation values are 1.40 and 4.67 microg/spot, respectively. Statistical analysis proves that the method is repeatable and accurate for the determination of RBV in the presence of its degradation products. The method meets the International Conference on Harmonisation/Food and Drug Administration regulatory requirements. The proposed TLC method is successfully applied for the determination of RBV, pure and in capsules, with good accuracy and precision; the label claim percentages are 98.8%+/-1.5%. The results obtained by the proposed TLC method are comparable with those obtained by the official method.     

Effective separation and simultaneous analysis of anabolic androgenic steroids (AAS) in their pharmaceutical formulations by a validated TLC-densitometry method

ABSTRACT: A TLC densitometric method was developed for simultaneous determination of four anabolic androgenic steroids (AAS) of testosterone derivatives including testosterone propionate (TP), testosterone phenyl propionate (TPP), testosterone isocaproate (TI) and testosterone deaconate (TD) in their pharmaceutical products. Separation was carried out on Al based TLC plates, pre-coated with silica gel 60F-254 using hexane and ethyl acetate (8.5:1.5, v/v). Spots at Rf 0.31+/-0.01, 0.34+/-0.01, 0.40+/-0.01 and 0.45+/-0.02 were recognized as TPP, TP, TI and TD, respectively. Quantitative analysis was done by densitometric measurements at lambdamax 251 nm for all derivatives. The developed method was validated as per ICH guidelines. Method was found linear over the concentration range of 200-1200 ng/spot with the correlation coefficient of 0.995, 0.993, 0.995 and 0.996 for TP, TPP, TI, TD, respectively. Limit of detection for all derivatives were in the range of 16.7-22.3 ng/spot while limit of quantitation were found to be in the range of 55.7-70.9 ng/spot. The developed TLC method can be applied for the simultaneous routine analysis of testosterone derivatives in their individual and combined pharmaceutical formulations.     

Estimation of berberine in ayurvedic formulations containing Berberis aristata

A sensitive, simple, rapid, and efficient high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the analysis of berberine in marketed Ayurvedic formulations containing Berberis aristata DC for regulatory purposes. Chromatography of methanolic extracts of these formulations was performed on silica gel 60 F254 aluminum-backed TLC plates of 0.2 mm layer thickness. The plate was developed up to 66 mm with the ternary-mobile phase butanol-acetic acid-water (8 + 1 + 1, v/v/v) at 33 +/- 5 degrees C with 5 min of tank saturation. The marker, berberine, was quantified at its maximum absorbance of 350 nm. The limit of detection and limit of quantitation values were found to be 5 and 10 ng/spot. The linear regression analysis data for the calibration plot showed a good linear relationship with correlation coefficient = 0.9994 in the concentration range of 10 to 50 ng/spot for berberine with respect to peak area. The instrumental precision was found to be 0.49% coefficient of variation (CV), and repeatability of the method was 0.73% CV. Recovery values from 98.27 to 99.11% indicate excellent accuracy of the method. The developed HPTLC method is very accurate, precise, and cost-effective, and it has been successfully applied to the assay of marketed formulations containing B. aristata for determination of berberine.     

超高效液相色谱-串联质谱法测定浓缩苹果汁中的熊果苷

建立浓缩苹果汁样品中熊果苷的固相萃取-超高效液相色谱-串联质谱(SPE-UPLC-MS/MS)检测方法。浓缩苹果汁样品用水溶解、过滤后,用聚苯乙烯-二乙烯基苯共聚物(PS-DVB)固相萃取柱净化,外标法定量。测定时用Eclipse Plus C18色谱柱(100 mm×2.1 mm, 1.8 μm)分离,甲醇-水系统梯度洗脱;MS测定采用多反应监测(MRM)模式。熊果苷的检出限为0.02 mg/L,在0.04～2.0 mg/L的范围内标准溶液的峰面积与质量浓度呈良好的线性关系,回收率为75.2%～102.7%,相对标准偏差(RSD)低于8.9%。该方法简便、快速、灵敏,可用于浓缩苹果汁样品中熊果苷的检测和确证。     

Quantitative determination of clozapine in serum by instrumental planar chromatography

An instrumental planar chromatographic (HPTLC) method for quantitative analysis of clozapine in human serum was developed and validated. Clozapine was extracted with n-hexane-isoamyl alcohol (75:25 v/v). The chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of chloroform and methanol (9:1 v/v) as mobile phase. Quantitative analyses were carried out by densitometry at a wavelength of 290 nm. The method was linear between 10 and 100 ng/spot, corresponding to 0.10 and 1.00 ng/microL of clozapine in human serum after extraction process and applying 10 microL to the chromatographic plates. The method correlation coefficient was 0.999. The intra-assay variation was between 2.10 and 3.33% (n = 5) and the interassay was between 2.67 and 4.44% (n = 9). The detection limit was 0.03 ng/microL, and the quantification limit was 0.05 ng/microL. The method proved to be accurate, with a recovery between 97.00 and 99.00%, with an RSD not higher than 7.22%, and was selective for the active principle tested. This method was successfully applied to quantify clozapine in patient serum samples. In conclusion, the method is useful for the quantitative determination of clozapine in serum.     

High-performance liquid chromatography and thin-layer chromatography for the simultaneous quantitation of rabeprazole and mosapride in pharmaceutical products

Simple, sensitive high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) methods are developed for the quantitative estimation of rabeprazole and mosapride in their combined pharmaceutical dosage forms. In HPLC, rabeprazole and mosapride are chromatographed using 0.01M 6.5 pH ammonium acetate buffer-methanol-acetonitrile (40:20:40, v/v, pH 5.70+/-0.02) as the mobile phase at a flow rate of 1.0 mL/min. In TLC, the mobile phase is ethyl acetate-methanol-benzene (2:0.5:2.5, v/v). Both the drugs are scanned at 276 nm. The retention times of rabeprazole and mosapride are found to be 4.93+/-0.01 and 9.79+/-0.02, respectively. The Rf values of rabeprazole and mosapride are found to be 0.42+/-0.02 and 0.61+/-0.02, respectively. The linearities of rabeprazole and mosapride are in the range of 400-2000 ng/mL and 300-1500 ng/mL, respectively, for HPLC; in TLC, the linearities of rabeprazole and mosapride are in the range of 400-1200 ng/spot and 300-900 ng/spot, respectively. The limit of detection is found to be 97.7 ng/mL for rabeprazole and 97.6 ng/mL for mosapride in HPLC; in TLC the limit of detection is found to be 132.29 ng/spot for rabeprazole and 98.25 ng/spot for mosapride. The proposed methods can be applied to the determination of rabeprazole and mosapride in combined pharmaceutical products.     

Validated HPLC Method for Simultaneous Quantitation of Bergenin,Arbutin, and Gallic Acid in Leaves of Different Bergenia Species

Bergenia species (Saxifragaceae) are important sources of herbal medicines in Asia, mainly in Russia. Various plant parts are valued for their antibacterial, anti-inflammatory, antioxidant sand adaptogenic effect, and used for the dissolution of kidney and bladder stones. In this study a rapid reversed phase liquid chromatography (RP-HPLC) method has been developed for rapid screening and identifying of the main active components in leaf samples of Bergenia accessions. The main goal of this study was to develop an efficient method for the simultaneous identification and detection of arbutin, bergenin and gallic acid from Bergenia leaf samples, which were extracted with a methanolic solvent mixture [methanol:water = 1:1 (v/v)]. Chromatographic separations were performed on a reversed phase Luna C18(2)-HST HPLC column. This chromatographic system provided increased speed and efficiency for separations, without the need for ultra-high pressures. Reversed phase HPLC coupled with diode array detector method was used for the analysis. The method was validated using ICH guidelines. The level of gallic acid was significantly higher in Bergenia crassifolia samples compared to Bergenia cordifolia. However, the samples of the two Bergenia species did not differ substantially regarding the concentrations of arbutin and bergenin. The novel method proved to be fast and allowed sufficient separation and quantification of arbutin, bergenin and gallic acid, the most important bioactive compounds of Bergenia leaves; thus facilitating rapid screening and quality assessment of Bergenia samples of various botanical and geographical origins.

     

Stability-indicating high performance thin layer chromatographic determination of sulfanilamide in human urine

A simple, sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method was developed and applied to human urine for the densitometric determination of sulfanilamide. A mixture of chloroform-ethyl acetate-xylene (2.5: 4.0: 1.0, v/v/v) was used as a mobile phase. The system was found to give compact spots for sulfanilamide (retardation factor, R _f = 0.21±0.02). The linear regression analysis data for the calibration plots showed good linear relationship with r ² = 0.9970 ± 0.0003 and r ² = 0.9947 ± 0.020 within the concentration range of 50–250 ng per spot and 100–1000 ng per spot with respect to peak area, respectively. The limit of detection (LOD) and quantification (LOQ) were 8 and 25 ng per spot, respectively. Sulfanilamide was subjected to acid and alkali hydrolysis, oxidation, dry heat and wet heat treatment. According to the International Conference on Harmonization (ICH) guidelines the method was validated for precision, recovery and robustness. The ultraviolet (UV) spectra of the degradation products which had different spectra from sulfanilamide were also recorded. The article is published in the original.     

Simultaneous densitometric analysis of amlodipine,hydrochlorothiazide, lisinopril,and valsartan by HPTLC in pharmaceutical formulations and human plasma

A simple, accurate, and precise high-performance thin layer chromatographic (HPTLC) method has been developed and validated for the simultaneous quantification of antihypertensive drugs, amlodipine (AML), hydrochlorothiazide (HCTZ), lisinopril (LIS), and valsartan (VAL) in their pharmaceutical formulations and human plasma. Separation of the drugs was performed on aluminum-backed layer of silica gel 60?F₂₅₄ using a mixture of methanol–dichloromethane–glacial acetic acid (9.0:1.0:0.1, v/v/v) as the mobile phase. Densitometric determination of the separated spots was done at 215?nm. The retention factors (R_f) obtained under the optimized conditions were 0.56, 0.75, 0.29, and 0.67 for AML, HCTZ, LIS, and VAL, respectively. Linearity of the method was established in the range of 200–1,500?ng/band for AML, 300–1,500?ng/band for HCTZ, 400–2,000?ng/band for LIS, and 1,000–7,000?ng/band for VAL. The limit of detection/limit of quantitation of the method found were 54.21/164.28, 77.27/234.15, 83.45/252.87, and 156.48/474.19?ng/band for AML, HCTZ, LIS, and VAL, respectively. To determine the drugs in spiked plasma samples, solid phase extraction was performed, which provided highly consistent and quantitative recovery for all four drugs. The method was satisfactorily applied for the analysis of different tablet formulations and proved to be specific and accurate for the quality control of these drugs.     

Thin-layer chromatography-densitometric measurements for determination of N-(hydroxymethyl)nicotinamide in tablets and stability evaluation in solutions

A thin-layer chromatography (TLC)-densitometric method was developed to determine N-(hydroxymethyl)nicotinamide in tablets and basic solutions along with nicotinic acid. Analysis was performed on silica gel F254 plates using chloroform-ethanol (2 + 3, v/v) mobile phase. The densitometric observations were made at 260 nm. The results showed good precision and accuracy; relative standard deviation was 2.37%, and recovery ranged from 97.60 to 100.82%. The limit of detection was 0.1 microg/spot, while the linearity range was from 0.2 to 1.75 microg/spot. Applicability of the newly developed method was tested for determination of N-(hydroxymethyl)nicotinamide in the preparation Cholamid. Densitometric measurements were used to evaluate stability of N-(hydroxymethyl)nicotinamide in basic solutions. It was found that decomposition corresponded to first-order reaction kinetics. The computed kinetic and thermodynamic parameters at 30 degrees C were as follows: k = 0.00675/min, t0,5 = 1.71 h, t0,1 = 0.26 h, and Ea = 44.75 kJ/mol.     

Detection of antioxidant butylated hydroxytoluene (BHT) in Antarctic krill (Euphausia superba Dana)

In this study, butylated hydroxytoluene (BHT) was separated and prepared from Antarctic krill. BHT was separated from the volatile oil of Antarctic krill using high performance thin layer chromatography (HPTLC) with petroleum ether/ethyl acetate/hexane (4:1:0.5, v/v/v) was used as the developing solvent. The content of BHT in volatile oil was 9.20?mg/g and the content of BHT in dried Antarctic krill was 0.35?mg/g (0.070?mg/g in frozen whole Antarctic krill). The linearity, accuracy, stability, and recovery of the analysis showed that HPTLC is the most suitable method for the determination of BHT in Antarctic krill. The BHT crude sample was obtained by scraping the separated spot in the thin layer chromatography plate, which was then analyzed using high performance liquid chromatography (HPLC). The resulting sample was identified with 96.05% purity based on the HPLC analysis. The structure of BHT was determined using gas chromatography–mass spectrometry (GC–MS). The results of the HPLC and GC–MS analysis validated the HPTLC method. BHT is a widely used antioxidant in food, pharmaceuticals, and in industrial production. The exploitation and utilization of BHT in Antarctic krill is of great economic value.     

The ICH guidance in practice: stress degradation studies on stavudine and development of a validated specific stability-indicating HPTLC assay method

A sensitive, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for the analysis of stavudine both as a bulk drug and in formulations is developed and validated. The solvent system consisted of toluene-methanol-chloroform-acetone (7.0:3.0:1.0:1.0, v/v/v/v). Densitometric analysis of stavudine is carried out in the absorbance mode at 270 nm. This system is found to give compact spots for stavudine (retention factor value of 0.45 +/- 0.05) following development of chromatoplates with the mobile phase. Stavudine is subjected to acid and alkali hydrolysis, oxidation, dry-heat and wet-heat treatment, and photo and UV degradation. The drug undergoes degradation under acidic and basic conditions, oxidation, and wet-heat degradation. Linearity is found to be in the range of 30-1000 ng/spot with a significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots show a good linear relationship with r2 = 0.9997 +/- 0.05 in the working concentration range of 300 to 1000 ng/spot. The mean value of slope and intercept are 0.10 +/- 0.06 and 22.12 +/- 1.08, respectively. The method is validated for precision, robustness, and recovery. The limits of detection and quantitation are 10 and 30 ng/spot, respectively. The proposed HPTLC method is utilized to investigate the kinetics of the acid degradation process. Arrhenius plot is constructed and activation energy is calculated.     

Indirect spectrophotometric determination of arbutin, whitening agent through oxidation by periodate and complexation with ferric chloride

A simple and accurate spectrophotometric method for the determination of arbutin (glycosylated hydroquinone) is described. It is based on the oxidation of arbutin by periodate in presence of iodate. Excess periodate causes liberation of iodine at pH 8.0. The unreacted periodate is determined by measurement of the liberated iodine spectrophotometrically in the wavelength range (300-500 nm). A calibration curve was constructed for more accurate results and the correlation coefficient of linear regression analysis was -0.9778. The precision of this method was better than 6.17% R.S.D. (n=3). Regression analysis of Bear-Lambert plot shows good correlation in the concentration range 25-125 ug/ml. The identification limit was determined to be 25 ug/ml a detailed study of the reaction conditions was carried out, including effect of changing pH, time, temperature and volume of periodate. Analyzing pure and authentic samples containing arbutin tested the validity of the proposed method which has an average percent recovery of 100.86%. An alternative method is also proposed which involves a complexation reaction between arbutin and ferric chloride solution. The produced complex which is yellowish-green in color was determined spectophotometrically.     

Validated HPLC Method for Simultaneous Quantitation of Bergenin,Arbutin, and Gallic Acid in Leaves of Different Bergenia Species

Bergenia species (Saxifragaceae) are important sources of herbal medicines in Asia, mainly in Russia. Various plant parts are valued for their antibacterial, anti-inflammatory, antioxidant sand adaptogenic effect, and used for the dissolution of kidney and bladder stones. In this study a rapid reversed phase liquid chromatography (RP-HPLC) method has been developed for rapid screening and identifying of the main active components in leaf samples of Bergenia accessions. The main goal of this study was to develop an efficient method for the simultaneous identification and detection of arbutin, bergenin and gallic acid from Bergenia leaf samples, which were extracted with a methanolic solvent mixture [methanol:water = 1:1 (v/v)]. Chromatographic separations were performed on a reversed phase Luna C18(2)-HST HPLC column. This chromatographic system provided increased speed and efficiency for separations, without the need for ultra-high pressures. Reversed phase HPLC coupled with diode array detector method was used for the analysis. The method was validated using ICH guidelines. The level of gallic acid was significantly higher in Bergenia crassifolia samples compared to Bergenia cordifolia. However, the samples of the two Bergenia species did not differ substantially regarding the concentrations of arbutin and bergenin. The novel method proved to be fast and allowed sufficient separation and quantification of arbutin, bergenin and gallic acid, the most important bioactive compounds of Bergenia leaves; thus facilitating rapid screening and quality assessment of Bergenia samples of various botanical and geographical origins.     

Applying green analytical chemistry for development and validation of RP-HPLC stability indicating assay method for estimation of fenoverine in bulk and dosage form using quality by design approach

A green and robust reverse-phase liquid chromatographic method has been developed for the determination of fenoverine (FEN), by applying combined principles of green analytical chemistry and quality by design approaches on a Spherisorb C18 column (150?×?4.6?mm, 3?µm) with UV detection at 262?nm. A two level fractional factorial design (2^^7-3) Res IV was used for screening of influential chromatographic factors. The critical method parameters actively affecting critical quality attributes (CQAs) were identified and further optimized using Box–Behnken design. The predicted optimum assay conditions comprised of methanol and ammonium acetate buffer 20?mM, in an extent of 81:19% v/v individually having a flow rate of 1.0?mL/min with a column oven temperature of 33°C. The drug was stressed in hydrolytic, oxidative, reductive, thermal, and photolytic conditions. The developed method was validated successfully. The detector response was linear in the concentration of 0.5–160?µg/mL with a limit of detection (LOD) and limit of quantitation (LOQ) as 0.1 and 0.3?µg/mL, respectively. The % recovery was found to be 99.7%. The analytical method volume intensity value for developed method was 45?mL and the environment assessment tool (EAT) score was 41.07. The method is simple, environmentally benign, rapid, and robust for the determination of FEN in bulk and in its dosage form.     

Separation and determination of rofecoxib and its degradation products by TLC chromatography

A thin layer chromatography with densitometry method for the determination of rofecoxib and degradation products is described. The chromatographic separation was performed on silica gel TLC plates as a stationary phase and chloroform-acetone-toluene-glacial acetic acid (12: 5: 2: 0.1, v/v/v/v) as a mobile phase. Densitimetric detection was carried out at 256 nm. The method is of high sensitivity and low LOD and LOQ: from 0.35 μg/spot to 1.05 μg/spot. The recovery was satisfactory at 98.62%. In addition, the stability of rofecoxib in solutions was investigated, including an effect of solution pH, temperature and incubation time. The method is rapid, easy and selective, particularly for the analysis of rofecoxib formulations. The article is published in the original.     

Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC–fluorimetry

Thiamine deficiency, if detected early in infancy, can be treated with thiamine supplementation and can prevent seizures, other disabilities and death. The dried blood spot (DBS) sampling technique is an attractive sample collection technique for infants. The present study reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from DBS. The method utilizes full‐spot analysis of a volumetrically deposited 40 μl DBS. The analyte was extracted from the DBS using 50% methanol and then derivatized using potassium ferricyanide to thiochrome. Separation was achieved with the help of an Inertsil ODS C₁₈ column (5.0 μm, 250 × 4.6 mm) using 150 mm phosphate buffer pH 7–acetonitrile (90:10, % v/v) as the mobile phase. The use of a fluorimetric detector gave a good response to the thiochrome derivative offering good sensitivity for the method. The excitation and emission wavelengths were 367 and 435 nm, respectively. The limit of detection and lower limit of quantification were 5 and 10 ng/ml, respectively. Linearity was demonstrated from 10 to 1000 ng/ml, and precision (CV) was <12.08%, at all tested quality control levels. The method accuracy was 89.34–118.89% with recoveries >80%. Bland–Altman analysis of DBS sampling vs. whole blood demonstrated a mean bias of only 1.16 ng/ml, with a majority of the 60 investigated patient samples lying within 7.2% of the corresponding concentration measured in blood, thereby meeting the clinical desirable biological specification criterion and showing that the two methods are comparable.