Normal-phase High-performance Liquid Chromatographic Method Research Articles

Fast Determination of Fat-soluble Vitamins in Pharmaceutical Preparations by High-Performance Liquid Chromatography

Background: Vitamins are needed for the healthy functioning of the body. When these vitamins are not in sufficient amounts in the body, they are usually taken externally with pharmaceutical preparations. Taking vitamins into the body in the right amounts is possible by analyzing the amounts in pharmaceutical preparations with validated methods. Methods: In this study, a fast, simple, easily applicable, and selective normal-phase HPLC method was developed for the simultaneous determination of fat-soluble A, D3, E, and K1 vitamins in pharmaceutical preparations from syrup and tablets. Separation of the vitamins was carried out on a Zorbax CN column (250 x 4.6 mm, 5 µm) using a mixture of hexane-isopropyl alcohol (98:2, v/v) at 30°C column temperature and 1 ml/min flow rate. The detection wavelength is 280 nm. The developed method has been validated according to ICH Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2(R1) rules. Results: Calibration graphs are linear over the range of 10-1000 µg ml-1 , 1-50 µg ml-1 , 10-2000 µg ml-1 , 0.5-20 µg ml-1, and the limit of detection values were found to be 1.496, 0.280, 1.388 and 0.040 µg ml-1 for A, D3, E and K1 vitamins, respectively. Relative standard deviation values, which express within-day and between-day repeatability, were found below 2.54%. Average recovery values were also found at about 100.28, 101.46, 100.65, and 100.29% for A, D3, E, and K1 vitamins, respectively Conclusion: The developed and validated method was successfully applied to the simultaneous analysis of fat-soluble vitamins A, D3, E, and K1 in pharmaceutical preparations in syrup and tablet form.

Separation and quantitative estimation of stereo-selective enantiomers of montelukast in pharmaceutical drug substance and tablets dosage forms by using stability-indicating normal phase-HPLC method.

Montelukast sodium (MLS) is a leukotriene receptor antagonist that relieves asthma, bronchospasm, allergic rhinitis, and urticaria. A simple, robust, and stability-indicating normal phase high-performance liquid chromatography method was developed to separate and quantitatively estimate the S-enantiomer of MLS. The chiral separation was achieved using USP L51 packing material along with a mobile phase consisting of a solvent mixture (n-hexane, ethanol, and propionic acid), a flow rate of 1.0mL/min, a detection wavelength of 284 nm, a column temperature of 30°C and an injection volume of 20 μL. The enantiomers peaks were well separated from the peaks of the placebo, diluting solvent, MLS, and its known impurities with a resolution of more than 2.2 and with no interference. Accuracy and linearity were studied in a range of 0.36-3.597 μg/mL (0.03%-0.30%), with good recoveries between 92.5% and 96.8% and a linear regression coefficient above 0.996. The suggested chiral chromatography method is being considered as an alternative and equivalent method to the United States Pharmacopeia and European Pharmacopeia monographs. The developed method was effectively employed for the study of release and stability samples of MLS. This HPLC method is also capable of separating and estimating the stereo-selective isomers (R- and S-enantiomers) of sulfoxide impurity of MLS in pharmaceutical medicine.

Stability-indicating normal-phase HPLC method development for separation and quantitative estimation of S-enantiomer of lacosamide in pharmaceutical drug substance and tablet dosage form.

Lacosamide (LA) is an antiepileptic medicine that is used to treat tonic-clonic seizures, partial-onset seizures, mental problems, and pain. A simple, effective, and reliable normal-phase liquid chromatographic technique was developed and validated to separate and estimate the enantiomer of (S-enantiomer) LA in pharmaceutical drug substance and drug product. Normal-phase LC was performed using USP L40 packing material (250 × 4.6mm, 5μm) and a mobile phase of n-hexane and ethanol at 1.0ml min-1 . The detection wavelength, column temperature, and injection volume used were 210 nm, 25°C, and 20 μl, respectively. The enantiomers (LA and S-enantiomer) were completely separated using a minimum resolution of 5.8 and accurately quantified without any interference in a 25-min run time. Accuracy study for stereoselective and enantiomeric purity trials was conducted between 10 and 200%, with recovery values ranging from 99.4 to 103.1% and linear regression values >0.997. The stability-indicating characteristics were assessed using forced degradation tests. The proposed normal-phase HPLC technique is an alternate approach to the official monograph methods (USP and Ph.Eur.) of LA, and it was successfully used in the evaluation of release and stability samples for both tablet dosage forms and pharmaceutical substances.

A new isoflavone from Dalbergia odorifera and inhibitory activity of its tyrosinase

Twelve flavonoids were isolated and purified from the ethyl acetate fraction of 95% ethanol extract of Dalbergia odorifera by heat reflux extraction, solvent extraction, recrystallization, normal phase silica gel, Sephadex LH-20, MCI gel and HPLC methods. The structures were identified with multiple spectroscopic methods, including 1 D-NMR, 2 D-NMR and MS. The compounds were identified as 6,7,8-trimethoxy-5,4'-dihydroxy isoflavone(1), medicarpin(2), 7,2'-dihydroxy-4'-methoxy-isoflavanol(3), biochanin A(4), prunetin(5), genistein(6), pratensein(7), 3-(4-hydroxyphenyl)-6-isopentenyl-7-methoxy-4H-chromen-4-one(8), tectorigenin(9), irisolidone(10), vestitol(11), and formononetin(12). Compound 1 was a new isoflavone, and compound 8 was isolated from D. odorifera for the first time. The results showed that compounds 1-3 had inhibitory effects on tyrosinase, with inhibition rates of 35.58%, 38.63% and 51.34% at the concentration of 1.0 mmol·L~(-1), respectively.

Determination of malathion and its residues by normal-phase high-performance liquid chromatography method

Abstract The quality of pesticide formulations has an impact on the crop safety, environment and human health. Therefore, the development of new analytical methods for the determination of active substances in pesticide formulations in order to control their quality, as well as, their residues in food samples in order to ensure food safety, is always welcome. A new, simple, precise and accurate normal-phase high-performance liquid chromatography (NP-HPLC) method for determination of an active ingredient malathion in the commercial emulsifiable concentrate pesticide product has been developed and validated. The analysis was carried out on a LiChrosorb CN (250 x 4 mm, 5 μm) analytical column using isocratic elution with mobile phase consisted of n-hexane and dichloromethane (80/20, v/v), flow rate of 1 mL/min, constant column temperature at 25 °C and ultraviolet diode-array detection at 220 nm. The obtained values for multiple correlation coefficients (R 2 ≥ 0.9990), relative standard deviation of retention times, peak areas and heights (RSD ≤ 1.14%), recoveries ranged from 98.97 to 101.62%, revealed that the developed method has a satisfactory linearity, precision and accuracy. Also, the developed method was successfully applied for determination of malathion residues in apple juice samples, after preliminary sample preparation using solid-phase extraction. Specificity, selectivity, linearity, matrix effect, precision and accuracy were tested in order to validation of this method. The obtained results were in acceptable ranges and indicated that the developed method is suitable for routine determination of malathion in the pesticide formulation, as well as for determination of malathion residues in apple juice samples. The run time of HPLC analysis was about 6 min.

Analytical developments of p-hydroxy prenylamine reference material for dope control research: Characterization and purity assessment.

Prenylamine was initially used for the treatment of angina pectoris and later on withdrawn from the market in 1988 due to cardiac arrhythmias concern. The major phase I metabolite of prenylamine is p-hydroxy prenylamine that has a chiral center in the structure. Even though p-hydroxy prenylamine was synthesized earlier, it lacked complete analytical developments for chiral high-performance liquid chromatography (HPLC) separation. However, p-hydroxy prenylamine reference material is not commercially available. The innovation of this manuscript is the development and validation of a chiral HPLC separation method and more extensive characterization of the reference material than previously reported method. Therefore, it was hypothesized to develop and validate normal phase HPLC method for p-hydroxy prenylamine reference material. p-Hydroxy prenylamine was synthesized in two batches and characterized successfully using 13 C NMR, 1 H NMR, high-resolution mass spectrometry (HRMS), Fourier transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA). A normal phase chiral HPLC method was developed to analyze the p-hydroxy prenylamine purity. Separation of the p-hydroxy prenylamine enantiomers were achieved using ultra-high-performance liquid chromatography (UHPLC) on a ChiralCel ODH column at wavelength of 220 nm. The developed method was validated in terms of its linearity, accuracy, precision, and robustness for purification, purity assessment, and stability studies. Proton and carbon peaks were confirmed by nuclear magnetic resonance (NMR) analysis. Functional groups were confirmed by FT-IR. Loss on drying was 0.3% and 0.6% for Batches 1 and 2, respectively. The purity of the developed reference material for Batches 1 and 2 was found to be 99.59% and 100%, respectively. Therefore, the synthesized batches of p-hydroxy prenylamine can be used in dope testing as reference material.

Determination of enantiomer impurity in Bortezomib lyo injection formulation by using normal-phase liquid chromatography

BackgroundA highly stereo-specific liquid chromatographic technique was built up and authenticated to quantify the (1S,2R-enantiomer) impurity in Bortezomib lyo injection formulation. The separation was achieved on Chiral Pak ID-3 (3 μm, 4.6 × 250 mm) column (“amylose-based 3-chlorophenylcarbamate” chiral stationary phase) through a movable segment consisting of n-heptane, 2-propanol, ethyl alcohol, and TFA (82:15:3:0.1, v/v/v/v) at a flow rate of 0.6 mL/min. Column temperature preserved 25 °C, injection level 20 μL, sample cooler temperature ambient, and detection wavelength 270 nm.ResultsThe retention time of (1S,2R-enantiomer) impurity and Bortezomib was determined 10.57 and 17.98 min, respectively. The resolution between (1S,2R-enantiomer) impurity and Bortezomib was found to be 4.2. The acceptance limit of the (1S,2R-enantiomer) impurity is 0.5%. The established method was authenticated as per ICH guidelines in respect of precision, accuracy, sensitivity, linearity, specificity, ruggedness, and robustness. The minimum quantity of the sample required for detection (LOD) was observed at 0.282 μg per mL and similarly the quantifying sample (LOQ) was observed to be 0.896 μg per mL.ConclusionThe proposed normal phase-HPLC method that can quantify (1S,2R-enantiomer) impurity in Bortezomib lyo injection formulation at trace level concentration has been urbanized and authenticated as per ICH guidelines. The effectiveness of the technique was ensured by the specificity, exactitude, linearity, and accuracy. Hence, the method well suit for their intended purposes and can be successfully useful for regular analysis in laboratories and is suitable for the quality control.

Effects of Processing Time and Temperature on Flavanol and Procyanidin, Proanthocyanidin and Antioxidant Activity of Cocoa Bean in Taiwan

Flavanol in cocoa is beneficial to cardiovascular health. This study investigated the effect of roasting conditions, temperature ranging from 110 to 150°C and time duration 15-35 min, on the level of flavanol and procyanidin (FP), total proanthocyanidin (PAC) and antioxidant activity of fermented cocoa beans in Southern Taiwan. The content of FP in unroasted and roasted cocoa bean was determined by the normal phase HPLC method and the antioxidant activity was assayed by DPPH and ABTS methods. High roasting temperature for long time decreased the content of FP and PAC in cocoa beans, where ranged from 55.30±1.95 mg (-)-epicatechin equivalents /g and 103.56±8.33mg procyanidin B1/g (at 110°C for 15 min) to 19.12±0.66 mg (-)-epicatechin equivalents /g and 51.58±0.86 mg procyanidin B1/g (at 150°C for 35 min), respectively. Meanwhile, as the roasting temperature rose, the DPPH and ABTS free radical scavenging ability of roasted cocoa bean extracts decreased gradually, varying from the maximum (IC50 = 104±2 µg/ml and 50.4±1.1mg TE/g) for unroasted bean to the minimum (IC50 = 144±4 µg/ml and 27.7±2.4 mg TE/g), and then slightly increased to IC50=134±4 µg/ml and 31.5±0.7 mg TE/g, respectively at most severe roasting treatment, showing the formation of melanoidins and polyphenols caused the synergistic effect, leading to the rise of the antioxidant activity. The first order kinetic reaction and the Arrhenius equation were applied to forecast the change of FP concentration during roasting. The activation energy of FP degradation is 11.2 kcal/mol. The obtained kinetic equation could be useful to predict the remaining amount of FP in cocoa beans under the designed experiment. The cocoa products with higher flavanol content and antioxidant activity could be possible by setting appropriate roasting conditions.

Separation of cis/trans Isomers of 4,5-Dihydroxy­imidazolidine-2-thione and 4,5-Dimethoxy­imidazolidine-2-thione by Aqueous Normal-Phase HPLC Mode

An aqueous normal-phase high-performance liquid chromatography method was developed to separate cis/trans isomers of 4,5-dihydroxy­imidazolidine-2-thione and 4,5-dimethoxy­imidazolidine-2-thione on a Luna HILIC 200 A column containing silica surface covered with cross-linked diol with an eluent acetonitrile/water in the ratio of 95:5 v/v. Changing of the analysis parameters leads to selectivity of cross-linked diol towards analyte with a predominance of adsorption mechanism between the phase and compounds. The separation in reversed-phase and normal-phase modes are unsuccessful. Chromatographic peaks of the cis/trans isomers are identified by the HR-LC–MS and the spectral ratios. The results of the determination of the isomer ratio for 4,5-dihydroxy­imidazolidine-2-thione by the HPLC are consistent with the 1H NMR data. The existence of the previously undescribed cis-4,5-dimethoxy­imidazolidine-2-thione is confirmed by the HR-HPLC–MS. This method allows providing simultaneous rapid separation and determination of the ratio of isomers in the samples of 4,5-dihydroxy­imidazolidine-2-thione and 4,5-dimethoxy­imidazolidine-2-thione.

Quantification of cranberry proanthocyanidins by normal-phase high-performance liquid chromatography using relative response factors.

American cranberries (Vaccinium macrocarpon) contain primarily A-type proanthocyanidins (PACs), which have been shown to prevent urinary tract infection. Currently, the accurate quantification of cranberry PACs is still lacking. A normal-phase high-performance liquid chromatography (NP-HPLC) method using relative response factors was developed and validated to quantify cranberry PAC oligomers and polymers. PAC oligomers with degree of polymerisation (DP) 3-9 and total polymers were isolated from the cranberry juice concentrate. Characterisation of the isolated PAC oligomers was performed by ultra-performance liquid chromatography-high resolution mass spectrometry. The relative response factors of oligomers from DP 2-9 and total polymers were determined against procyanidin A2. Method validation was conducted to assess limit of detection, limit of quantification, the linearity and working range, precision and accuracy. In addition, quantifications of PACs by NP-HPLC using relative response factors and two other commonly used methods were conducted in three cranberry food products. Cranberries PACs oligomers contained both A-type and B-type linkage, with epicatechin and epigallocatechin as basic units. Method validation results suggested this method is reliable and reproducible. Quantifications of PACs by NP-HPLC using relative response factors yielded higher values than that by the other two methods. A NP-HPLC method using the relative response factors was developed and validated. This method provides a more accurate approach in determining cranberry PACs. It can be used to quantify individual oligomers from DP 2-9, total polymers and total PACs in cranberries and cranberry products.

Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux

Three normal phase HPLC methods were produced to separate lipid classes on a PVA-Sil stationary phase including: 9 polar lipids (method 1); 13 combined polar and neutral lipids (method 2); and a combined method that further separates the neutral lipids into 2–4 subclasses based on the presence of fatty acids containing a polar functional group (e.g. hydroxyl) for a total of 20 lipid classes and subclasses separated in a single run (method 3). Polar lipids separated include: the phosphoglycerolipids PG, PE, PI, PS, PC and LPC; the galactoglycerolipids MGDG and DGDG; and a sulfoglycerolipid SQDG. Neutral lipids include TAG, DAG, and MAG classes and sub-classes containing 0–3, 0–2, and 0–1 hydroxy fatty acids, respectively. The hexane/isopropanol/methanol/aqueous system separates polar lipids without the use of chloroform such that it is suitable for radioactivity analysis by in-line flow scintillation counting. Each method was optimized using the natural lipid standards comprised of diverse molecular species that were detected by ELSD. All molecular species of each lipid class eluted together as single peak detected by ELSD. The methods were demonstrated to be suitable for resolving lipid extracts from animal, microbial, and plant sources as well as application to 14C based metabolic tracing of lipid metabolism in leaves and seeds.

A NOVEL HPLC METHOD FOR ESTIMATION OF GENOTOXIC IMPURITY 3-ACETAMIDOBENZENE 1, 2 DICARBOXYLIC ACID IN KEY STARTING MATERIAL 3-ACETAMIDOPTHALIC ANHYDRIDE OF APREMILAST API

3-Acetamidobenzene -1, 2-dicarboxylic acid is a potential genotoxic impurity which gets formed during synthesis of 3- acetamidopthalic anhydride, a Key Starting Material (KSM) for manufacturing of apremilast API. During handling upon exposure to moisture, the anhydride ring of KSM 3-acetamidopthalic anhydride opens to form the acid. Hence Reverse phase HPLC method is not a feasible and robust option for estimation of this impurity. To overcome these problems a normal phase HPLC method is developed and proposed in this research work. Immobilized Chiral pack IA column from Daicel is used for estimation. n-Hexane and isopropyl alcohol in 90:10 (v/v) ratio modified with 0.1% trifluroacetic acid is used as mobile phase. Method is validated as per ICH guidelines. Limit of Detection (LOD) and Limit of Quantification (LOQ) are found to be 0.47 ppm (0.0047%) and 1.42 ppm (0.0142%), respectively. The method is linear over LOQ to 150%. Recovery is within limits (80-120%). Method is robust for parameters like mobile phase composition, flow rate, wavelength changes and column oven temperature.

Stereoselective HPLC separation of alvimopan on cellulose-based immobilized polysaccharide as a chiral stationary phase.

Chiral separation by normal phase high performance liquid chromatography is one of the most powerful technique to quantify the chiral purity of the compounds. In this study, a novel, simple, and specific analytical method was proposed to ascertain the chiral purity of alvimopan (ALV). The normal phase HPLC method was developed based on cellulose tris (3,5-dichlorophenylcarbamate) stationary phase. The separation of ALV isomers achieved by using column CHIRALPAK IC (250×4.6mm, 5μm), mobile phase n-hexane: isopropyl alcohol: ethanol: diethylamine (650:200:150:5 v/v), column oven temperature 30°C, flow rate 1.0mLmin-1 , injection volume was 10μL, chromatographic response monitored at 273nm. The developed method was validated as per the ICH guidelines and found precise, accurate, and linear. The advantage of the method is a good separation of ALV isomers within 35minutes of the analysis time. Therefore, this method is suitable for routine determination of chiral purity of ALV active pharmaceutical ingredient.

Development and Validation of a New Simple HPLC Method for the Determination of 3-[4-(2-Methylpropyl)phenyl] Propanoic Acid as an Impurity of Ibuprofen and Ibuprofen Sodium

Ibuprofen sodium is the sodium salts form of ibuprofen which exhibits better effects than standard ibuprofen as an analgesic by inhibiting the cyclooxygenase enzyme system. 3-[4-(2-Methylpropyl)phenyl] propanoic acid as one of potential impurities of ibuprofen was determined by a gas chromatography method after the methylation reaction according to the European Pharmacopoeia. Now, a new simple normal phase high-performance liquid chromatography (NP-HPLC) method has been established to separate 3-[4-(2-methylpropyl)phenyl]propanoic acid from ibuprofen sodium as well as from ibuprofen. Separation was performed on an Ultimate Silica column (250 mm × 4.6 mm, 5 μm) using hexane, ethyl acetate and trifluoroacetate in the ratio (97:3:0.95, v/v/v) as mobile phase. The detection wavelength was performed at 264 nm. The NP-HPLC method could successfully separate 3-[4-(2-methylpropyl)phenyl]propanoic acid from the main peak within an analysis time of less than 20 min. Complementary of this work, a forced degradation study was carried out under specified conditions to investigate the chemical stability of ibuprofen sodium. The method was found to be precise, linear, accurate, sensitive, specific and robust according to the ICH guidelines.

NP-HPLC method for determining phytonadione and its impurities

ABSTRACTA normal-phase high-performance liquid chromatography method has been developed to separate vitamin cis-phytonadione (cis-K1), trans-phytonadione (trans-K1), trans-epoxyphytonadione (trans-EK1), menadione (K3), cyclo-menadione (CK3), hydroperoxy-phytonadione (K1OOH), and other impurities on a Zorbax RX-SIL column, characterized by very pure porous silica, low acidity, and low metal content. A dual-mode gradient elution was applied involving simultaneous changes in flow rate and mobile-phase composition. The present method was sensitive enough to detect trans-EK1 (limit of detection, LOD: 1.8 ng), K3 (LOD: 0.6 ng), CK3 (LOD: 1.2 ng), and K1OOH (LOD: 2.4 ng). A linear relationship between peak area and concentration was found for four compounds with correlation coefficient r exceeding 0.9994. The impurities (trans-EK1, K3, and K1OOH) were measured accurately (recovery rate 80–120%) and precisely (relative standard deviation less than 2%). Preparing samples under yellow light (≥520 nm), the K1OOH in the three commercial batches of K1 was no higher than 0.10% (w/w) and was measured with a reproducibility of 3.1% and with an intermediate precision of 15.8%. After validation, the developed method was applied for analyzing the impurities occurring in the commercial samples of synthetic K1 and proved to be suitable for routine quality control.

Comparison of liquid and supercritical fluid chromatography mobile phases for enantioselective separations on polysaccharide stationary phases

Analysis and production of enantiomerically pure compounds is a major topic of interest when active pharmaceutical ingredients are concerned. Enantioselective chromatography has become a favourite both at the analytical and preparative scales. High-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) are dominating the scene and are often seen as complementary techniques. Nowadays, for economic and ecologic reasons, SFC may be preferred over normal-phase HPLC (NPLC) as it allows significant reductions in solvent consumption. However, the transfer of NPLC methods to SFC is not always straightforward.In this study, we compare the retention of achiral molecules and separation of enantiomers under supercritical fluid (carbon dioxide with ethanol or isopropanol) and liquid normal-phase (heptane with ethanol or isopropanol) elution modes with polysaccharide stationary phases in order to explore the differences between the retention and enantioseparation properties between the two modes. Chemometric methods (namely quantitative structure-retention relationships and discriminant analysis) are employed to compare the results obtained on a large set of analytes (171 achiral probes and 97 racemates) and gain some understanding on the retention and separation mechanisms. The results indicate that, contrary to popular belief, carbon dioxide − solvent SFC mobile phases are often weaker eluents than liquid mobile phases. It appears that SFC and NPLC elution modes provide different retention mechanisms. While some enantioseparations are unaffected, facilitating the transfer between the two elution modes, other enantioseparations may be drastically different due to different types and strength of interactions contributing to enantioselectivity.

Improved Chiral Separation of (R,S)-Goitrin by SFC: An Application in Traditional Chinese Medicine

Like chemical drugs, research and development of herbal medicine also have a need to resolve enantiomers. To help illustrating the antiviral bioactivity of Isatidis Radix, a widely used traditional Chinese medicine (TCM), supercritical fluid chromatography (SFC) was used for analytical and preparative separation of (R,S)-goitrin, which was reported as the active ingredient of the herbal. Improved resolution was achieved on Chiralpak IC column, using acetonitrile as the organic modifier, representing a tenfold increase in speed, compared to the previous normal phase HPLC (NPLC) method. The newly developed chromatographic method was validated in terms of linearity, precision, limit of detection (LOD), and limit of quantitation (LOQ). Scale-up purification of (R)-goitrin and (S)-goitrin was performed on a preparative column with >90% total recovery. The absolute stereochemical assignment of the purified isomers was determined through optical rotation study. This attempt explored SFC's application in chiral research of traditional Chinese medicine.

Determination of Vitamin K1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Fluorescence Detection: Single-Laboratory Validation, First Action 2015.09.

This normal-phase HPLC method with postcolumn reduction and fluorescence detection allows for the quantitative determination of trans vitamin K1 in infant, pediatric, and adult nutritionals. Vitamin K1 is extracted from products with iso-octane after precipitation of proteins and release of lipids with methanol. Prepared samples are injected onto a silica HPLC column where cis and trans vitamin K1 are separated with an iso-octane-isopropanol mobile phase. The column eluent is mixed with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and vitamin K1 is reduced to a fluorescent derivative in a zinc reactor column. The resulting hydroquinone is then detected by fluorescence at an excitation wavelength of 245 nm and an emission wavelength of 440 nm. During a single-laboratory validation of this method, repeatability and intermediate precision ranged from 0.6 to 3.5% RSD and 1.1 to 6.0% RSD, respectively. Mean overspike recoveries ranged from 91.9 to 106%. The method demonstrated good linearity over a standard range of approximately 2-90 μg/L trans vitamin K1 with r2 averaging 0.99995 and average calibration errors of <1%. LOQ and LOD in ready-to-feed nutritionals were estimated to be 0.03 and 0.09 μg/100 g, respectively. The method met AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Standard Method Performance Requirements® and was approved as a first action method at the 2015 AOAC Mid-Year Meeting.

An isocratic normal-phase high-performance liquid chromatographic method for the simultaneous determination of benzoyl peroxide and benzoic acid in one pharmaceutical preparation and their stability in different solvents

A normal-phase high-performance liquid chromatographic method was developed for the simultaneous determination of benzoyl peroxide (BPO) and benzoic acid (BA). The compounds are separated on a normal phase column Eurospher-100, c18.4.6mmID. The mobile phase is methanol - water (65 + 35, v/v). Solutions are injected into the chromatographic system under isocratic conditions at a constant flow rate of 1 mL /min with UV detection at 240 nm. Analysis of stability samples in deferent solvents showed formation of BPO from BA when it dissolved in ether or methanol and very low degradation of BPO to BA when it dissolved in ether compare with when it dissolved in methanol

Determination of (R)-timolol in (S)-timolol maleate active pharmaceutical ingredient: Validation of a new supercritical fluid chromatography method with an established normal phase liquid chromatography method

An enantioselective supercritical fluid chromatography (SFC) method was developed and validated to meet the current European Pharmacopoeia requirements of a limit test for the determination of S-timolol maleate enantiomeric purity in timolol maleate drug substance. The developed method is presented as an alternative to the current normal phase high performance liquid chromatography (NP-HPLC) method described in the European Pharmacopoeia (Timolol Maleate Monograph). Using a 4.6mm×250mm Chiralcel OD-H (dp: 5μm) column and a mobile phase of (93:7) CO2/0.1% (v/v) TEA in MeOH delivered at 4.0mLmin−1 resolution of 2.0 was achieved within 5min, representing a 3-fold reduction in run-time and an 11-fold reduction in solvent consumption relative to the NP-HPLC method. Method robustness was examined by the variation of flow rate (±0.5mLmin−1), column temperature (±5°C) and column back-pressure (±10bar) and resolution was maintained at ≥1.9 in all cases. R-timolol was resolved from all potential impurities and the limit of detection was improved by increasing the sample concentration threefold compared to the NP-HPLC method such that the method could detect the R-timolol enantiomer at 0.5% (w/w) with respect to S-timolol maleate. Additional validation parameters demonstrated that the potential of the method to be used for routine release testing of timolol maleate raw material for drug product manufacturing in which the quantitation of R-timolol impurity in S-timolol maleate drug substance would be a requirement.