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Sildenafil is an inhibitor of the phosphodiesterase type 5 which is commonly adulterated in herbal health products. In this study, a rapid, sensitive and selective method using thin layer chromatography (TLC) combined with surface-enhanced Raman spectroscopy (SERS) was developed for identification of sildenafil adulteration in herbal drugs and dietary supplements. TLC separation method was developed and different SERS factors were investigated: nanosilver colloid preparation, concentration and volume, and coffee-ring effect (CRE) enhancement for SERS measurement. "Double CRE" - a newly observed effect that resulted in the redistribution of both silver nanoparticles and sildenafil molecules - was reported for the first time in TLC-SERS applications. This method presented an efficient TLC-SERS performance enhanced by the "hot spots" obtained under double CRE. The method was validated in terms of selectivity on three blank matrixes (capsule, granule, and herbal extract) and sensitivity with a limitation of detection (LOD) of 2 ng/spot for sildenafil. The validated method was implemented on 9 herbal products sold on the market as erectile dysfunction therapy. Two products were detected with sildenafil adulteration by the TLC-SERS method and confirmed by parallel LC-MS/MS analysis. These results exhibited the reliability and feasibility of the developed method in adulteration screening for sildenafil. On the other hand, the novel findings on double CRE provided extra information for CRE optimization in TLC-SERS applications.

The quality control of active pharmaceutical ingredients (APIs) is a very important aspect for drug products entering the market. However, also for the well-established drugs, there ought to be a state-of-the-art impurity control. Some of the pharmacopoeial tests for related substances still make use of thin layer chromatography, even though selectivity and sensitivity are suboptimal. Here, we report on the development of a new gradient high performance liquid chromatography (HPLC) method for dapsone in order to replace the currently described pharmacopoeial TLC method. The separation of all relevant components was achieved on a C18 stationary phase (Waters XTerra® RP18 5 μm 4.6 × 250 mm) using a water-acetonitrile gradient. A limit of detection (LOD) of 0.02% was registered for all specified impurities. Additionally, within this study an “impurity of an impurity” was identified by means of LC–MS/MS.

RP-18 TLC chromatography was used to evaluate the pharmacokinetic properties (volume of distribution, VD; plasma protein binding, %PPB; the ability to cross the blood-brain barrier expressed as log PS and log BB) of several cosmetic raw materials – sunscreen and preservatives. The majority of these compounds are intended for topical use on skin and their drug-likeness and the ability to cross biological barriers are undesired. The retention parameters RM0, S, PC1 and RM75 % obtained for mobile phases containing six organic modifiers (methanol, acetonitrile, THF, acetone, dioxane, DMF) were used as the sole descriptors or combined with calculated physicochemical properties (PSA, MW, VM) of studied compounds. The chromatographic parameters considered in this study are, generally speaking, good predictors of the compounds’ pharmacokinetic properties VD, %PPB and log PS. RM75 % and the novel parameters derived from it (RM75 %/MW and RM75 %/VM) can be considered time- and cost-effective alternatives to the chromatographic parameters obtained by extrapolation or interpolation methods. In the case of some pharmacokinetic properties investigated in this study additional descriptors (PSA) have a significant influence on the quality of correlations.

Increased antimicrobial resistance together with the lack of new antimicrobial drugs suggest on an urgent need for new therapeutics in this field. Vanilidene derivatives of Meldrum’s acid present one of the possible approaches. In this work lipophilicity of 13 vanilidene derivatives of Meldrum’s acid as well as their predicted antimicrobial activity towards several characteristic species has been evaluated. 10 vanilidene derivatives have been previously synthesized and 3 new compounds are synthetized afterwards following the same procedure. These selected 13 candidates were examined using thin layer chromatography in two different solvent systems. Gained retention parameters were a starting point for further Quantitative Structure Property Relationships (QSRR) studies in which minimum inhibitory concentration (MIC) for Candida albicans, Trichoderma viride, Penicillium italicum, Fuscarium oxysporum, Pseudomonas aeruginosa and Escherichia coli were determined. Statistically significant QSRR models were established and clustering of the compounds was performed with the help of principal component analysis (PCA) and hierarchical cluster analysis (HCA). Absorption, Distribution, Metabolism, and Excretion (ADME) properties of investigated molecules were subjected to sum of ranking differences (SRD) analysis in order to explore their pharmacokinetic properties. SRD analysis was also performed for the ranking of the established QSRR models. It was shown that compounds 6, 8 and 9 possess a significant antimicrobial activity, satisfied ADME properties and these candidates should be further optimized in order to utilize unexplored potential of Meldrum's acid in synthesis of novel antifungal compounds.

Conventional isolation and identification of active compounds from herbs have been extensively reported by using various chromatographic and spectroscopic techniques. However, how to quickly discover new bioactive ingredients from natural sources still remains a challenging task due to the interference of their similar structures or matrices. Here, we present a grand approach for rapid analysis, forecast and discovery of bioactive compounds from herbs based on a hyphenated strategy of thin layer chromatography and ratiometric surface-enhanced Raman spectroscopy. The performance of the hyphenated strategy is first evaluated by analyzing four protoberberine alkaloids, berberine (BER), coptisine (COP), palmatine (PAT) and jatrorrhizine (JAT), from a typical herb Coptidis Rhizoma as an example. It has been demonstrated that this coupling method can identify the four compounds by characteristic peaks at 728, 708, 736 and 732 cm−1, and especially discriminate BER and COP (with similar migration distances) by ratiometric Raman intensity (I708/I728). The corresponding limits of detection are 0.1, 0.05, 0.1 and 0.5 μM, respectively, which are about 1–2 orders of magnitude lower than those of direct observation method under 254 nm UV lamp. Based on these findings, the proposed method further guides forecast and discovery of unknown compounds from traditional Chinese herb Typhonii Rhizoma. Results infer that two trace alkaloids (BER and COP) from the n-butanol extract of Typhonii Rhizoma are found for the first time. Moreover, in vitro experiments manifest that BER can effectively decrease the viability of human glioma U87 cells by inducing cell cycle arrest in a concentration-dependent manner.

The chromatographic and lipophilicity characters of seven cephalosporins of different four classes (cephradine, cefaclor, cefprozil, cefixime, cefotaxime, ceftazidime and cefepime) were examined by salting out thin-layer chromatography (SOTLC). SOTLC using ammonium sulfate salt was employed to predict the lipophilicity of the proposed drugs via their retention behavior. The calculated RM0 values showed liner relationship with the molar concentration of ammonium sulfate in mobile phase in the range of 0.5–2.5 mol/L. Additionally, quantitative structure retention relationship (QSRR) was generated to figure out the relationship between the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins. Good correlations were found between the chromatographically obtained retention parameters (RM0 and C0) and some molecular descriptors of the examined drugs. Furthermore, an efficient QSAR model was carried out using the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins to predict minimum inhibitory concentration (MIC) and blood brain barrier (BBB) penetration of the examined drugs. The study was extended to separate and quantify the selected antibiotics in their pure forms and pharmaceutical formulations. Normal phase thin layer chromatographic (NP-TLC) method using a usable developing system of acetone: methanol: water: ammonium hydroxide: glacial acetic acid (90: 10: 18: 3: 2, by volume) was successfully applied to resolve the studied cephalosporins. Linearity was achieved in the range of 0.2–3 µg/mL for most of the studied antibiotics. The developed SOTLC method can be considered as a good start alternative to reversed phase thin layer chromatography (RP-TLC) for prediction of the lipophilic properties of examined cephalosporins. Moreover, the proposed NP-TLC densitometric method can be easily applied for quality control analysis of the chosen drugs and other structurally related components.

The objective of this paper is to build QSRR/QSAR model for predicting the blood-brain barrier (BBB) permeability. The obtained models are based on salting-out thin layer chromatography (SOTLC) constants and calculated molecular descriptors. Among chromatographic methods SOTLC was chosen, since the mobile phases are free of organic solvent. As consequences, there are less toxic, and have lower environmental impact compared to classical reserved phases liquid chromatography (RPLC). During the study three stationary phase silica gel, cellulose plates and neutral aluminum oxide were examined. The model set of solutes presents a wide range of log BB values, containing compounds which cross the BBB readily and molecules poorly distributed to the brain including drugs acting on the nervous system as well as peripheral acting drugs. Additionally, the comparison of three regression models: multiple linear regression (MLR), partial least-squares (PLS) and orthogonal partial least squares (OPLS) were performed. The designed QSRR/QSAR models could be useful to predict BBB of systematically synthesized newly compounds in the drug development pipeline and are attractive alternatives of time-consuming and demanding directed methods for log BB measurement. The study also shown that among several regression techniques, significant differences can be obtained in models performance, measured by R2 and Q2, hence it is strongly suggested to evaluate all available options as MLR, PLS and OPLS.

High-end analytical methods provide excellent data but may lack the robustness required in large analytical studies. In particular complex chemical matrices may cause difficulties and increase the need for extensive sample preparation. For screening of patients we thus developed a low-tech assay to monitor bradykinin degradation by serum proteases. The bradykinin concentration mirrors the activity of angiotensin-converting enzyme (ACE). Dabsylated bradykinin (DBK) and its labeled fragments DBK1-8 and DBK1-5 were visualized by thin-layer chromatography using only 3 μL of serum. Lower DBK1-5 levels indicated reduced ACE activity due to medication (ACE-inhibitors) or disease. Provided that purified DBK is available, the assay protocol itself is very simple and does not require any expensive high-end equipment.

Active pharmaceutical ingredients (API) embedded in the excipients of the formula can usually be unravelled by normal Raman spectroscopy (NRS). However, more and more drugs with low API content and/or low Raman scattering coefficient were insensitive to NRS analysis, which was for the first time defined as Low API-Signal Drugs (LASIDs) in this paper. The NRS spectra of these LASIDs were similar to their dominant excipients' profiles, such as lactose, starch, microcrystalline cellulose (MCC), etc., and were classified into three types as such. 21 out of 100 kinds of drugs were screened as LASIDs and characterized further by Raman microscopic mapping. Accordingly, we proposed a tailored solution to the qualitation and quantitation problem of these LASIDs, using surface-enhanced Raman spectroscopic (SERS) detection on the thin layer chromatographic (TLC) plate both in situ and after-separation. Experimental conditions and parameters including TLC support matrix, SERS substrate, detection mode, similarity threshold, internal standard, etc., were optimized. All LASIDs were satisfactorily identified and the quantitation results agreed well with those of high performance liquid chromatography (HPLC). For some structural analogues of LASIDs, although they presented highly similar SERS spectra and were tough to distinguish even with Raman microscopic mapping, they could be successfully discriminated from each other by coupling SERS (with portable Raman spectrometer) with TLC. These results demonstrated that the proposed solution could be employed to detect the LASIDs with high accuracy and cost-effectiveness.

Bioactive compounds from endophytic fungi exhibit diverse biological activities which include anticancer effect. Capitalising on the abundance of unexplored endophytes that reside within marine plants, this study assessed the anticancer potential of ethyl acetate endophytic fungal extracts (i.e. MBFT Tip 2.1, MBL 1.2, MBS 3.2, MKS 3 and MKS 3.1) derived from leaves, stem and fruits of marine plants that grow along Morib Beach, Malaysia. For identification of endophytic fungi, EF 4/ EF 3 and ITS 1/ ITS 4 PCR primer pairs were used to amplify the fungal 18S rDNA sequence and ITS region sequence, respectively. The resultant sequences were subjected to similarity search via the NCBI GenBank database. High-performance thin layer chromatography (HPTLC) hyphenated with bioassays was used to characterise the extracts in terms of their phytochemical profiles and bioactivity. Microchemical derivatisation was used to assess polyphenolic and phytosterol/ terpenoid content whereas biochemical derivatisation was used to establish antioxidant activities and α-amylase enzyme inhibition. The sulforhodamine B (SRB) assay was used to assess the anticancer effect of the extracts against HCT116 (a human colorectal cancer cell line). The present results indicated MBS 3.2 (Penicillium decumbens) as the most potent extract against HCT116 (IC50 = 0.16 μg/mL), approximately 3-times more potent than 5-flurouracil (IC50 = 0.46 μg/mL). Stepwise multiple regression method suggests that the anticancer effect of MBS 3.2 could be associated with high polyphenolic content and antioxidant potential. Nonlinear regression analysis confirmed that low to moderate α-amylase inhibition exhibits maximum anticancer activity. Current findings warrant further in-depth mechanistic studies.

Ceramides (CERs) play a major role in skin barrier function and direct replacement of depleted skin CERs, due to skin disorder or aging, has beneficial effects in improving skin barrier function and skin hydration. Though, plants are reliable source of CERs, absence of economical and effective method of hydrolysis to convert the dominant plant sphingolipid, glucosylceramides (GlcCERs), into CERs remains a challenge. This study aims at exploring alternative GlcCERs sources and chemical method of hydrolysis into CERs for dermal application. GlcCERs isolated from lupin bean (Lupinus albus), mung bean (Vigna radiate) and naked barley (Hordium vulgare) were identified using ultra high performance liquid chromatography hyphenated with atmospheric pressure chemical ionization - high resolution tandem mass spectrometer (UHPLC/APCI-HRMS/MS) and quantified with validated automated multiple development-high performance thin layer chromatography (AMD-HPTLC) method. Plant GlcCERs were hydrolyzed into CERs with mild acid hydrolysis (0.1 N HCl) after treating them with oxidizing agent, NaIO4, and reducing agent, NaBH4. GlcCERs with 4,8-sphingadienine, 8-sphingenine and 4-hydroxy-8-sphingenine sphingoid bases linked with C14 to C26 α-hydroxylated fatty acids (FAs) were identified. Single GlcCER (m/z 714.5520) was dominant in lupin and mung beans while five major GlcCERs species (m/z 714.5520, m/z 742.5829, m/z 770.6144, m/z 842.6719 and m/z 844.56875) were obtained from naked barley. The GlcCERs contents of the three plants were comparable. However, lupin bean contains predominantly (> 98 %) a single GlcCER (m/z 714.5520). Considering the affordability, GlcCER content and yield, lupin bean would be the preferred alternative commercial source of GlcCERs. CER species bearing 4,8-sphingadienine and 8-sphingenine sphingoid bases attached to C14 to 24 FAs were found after mild acid hydrolysis. CER species with m/z 552.4992 was the main component in the beans while CER with m/z 608.5613 was dominant in the naked barley. However, CERs with 4-hydroxy-8-sphingenine sphingoid base were not detected in UHPLC-HRMS/MS study suggesting that the method works for mainly GlcCERs carrying dihydroxy sphingoid bases. The method is economical and effective which potentiates the commercialization of plant CERs for dermal application.

Thin-layer chromatography (TLC) has a myriad of separation applications in chemistry, biology, and pharmacology due to its simplicity and low cost. While benchtop laboratory sample application and detection systems for TLC provide accurate quantitation of TLC spot positions and densities, there are many applications where inexpensive and portable instruments would greatly expand the applicability of the technology. In this work, we demonstrate identity verification and concentration determination of pharmaceutical compounds via TLC using a custom 3D-printed cradle that interfaces with an ordinary mobile phone. The cradle holds the mobile phone's internal, rear-facing camera in a fixed position relative to a UV lamp and a TLC plate that includes a phosphor in the stationary phase. Analysis of photographs thus reveals the locations and intensities of principal spots of UV--absorbing drugs. Automated image analysis software determines the center location and density of dark spots, which, using integrated calibration spots of known drug compounds and concentrations, can be used to determine if a drug has been diluted or substituted. Two independent image processing approaches have been developed that may be selected based upon the processing capabilities of the smartphone. Each approach is able to discern 5% drug concentration differences. Using single-component solutions of nevirapine, amodiaquine, and paracetamol that have been manually applied, the mobile phone-based detection instrument provides measurements that are equivalent to those obtained with a commercially available lab-based desktop TLC densitometer.

Two high performance chromatographic methods were developed and validated for the simultaneous determination of Ambroxol, Guaifenesin and Theophylline in pharmaceutical dosage forms and in the presence of Guaiacol and Caffeine as the officially stated impurities. These were a reversed phase liquid and a thin layer chromatographic methods. The liquid chromatographic separation was achieved using Inertsil ODS-3 C18 column (4.6 mm × 250 mm, 5 μm). Gradient elution was performed using a mixture of solvent A (0.05 M ammonium acetate, pH 3, adjusted with glacial acetic acid) and solvent B (methanol), at a flow rate of 1.0 mL/min. The separated peaks were detected at 260.0 nm. The thin layer chromatography was performed using HPTLC 60 F254 silica gel plates, mobile phase was consisting of ethyl acetate: methanol: acetic acid (10:0.5:1, v/v/v) and detection was performed at 254.0 nm. Validation of the developed methods was achieved according to International Conference on Harmonization (ICH) guidelines. The proposed methods were fast, accurate, precise, and sensitive. Hence, they could be employed for routine quality control of the ternary mixture in capsule and syrup dosage forms.

Chromatographic properties of sixteen β-blockers were studied using planar chromatography. The aim of presented study was to investigate influence of different organic solvents (acetonitrile, methanol, dioxin and tetrahydrofurane) on β-blockers’ retention on C18 bonded silica gel stationary phase. Group of sixteen, diverse in terms of structure, beta blockers was used as a model set. The main goal of this study was to compare chromatographically estimated lipophilicity parameters with values obtained with the use of computational methods. Furthermore, in order to understand molecular mechanisms of retention better, quantitative structure-retention relationships (QSRR) analysis was performed. The next step was focused on application of chromatographically obtained lipophilicity parameters for prediction of protein binding (PB), based on quantitative retention–activity relationships (QRAR) approach. The obtained results showed that reversed-phase thin layer chromatography (RP-TLC), especially with tetrafydrofurane used as an organic modifier of mobile phase, is a useful tool for lipophilicity estimation, as well as for prediction of β-blockers’ biological properties. The QRAR model included C0 parameters for tetrahydrofuran-water as a mobile phase, as well as maximal projection area, and can be easily applied for prediction of systematically synthesized β-blockers structures’ PB.

Thin layer chromatography in combination with image analysis and advanced chemometric methods were successfully used to classify the medicinal herbs according to their therapeutic effects and usage. The investigations were conducted using two types of plates (HPTLC Silica gel 60 and HPTLC Silica gel 60 F254) which were evaluated in UV light at 254 and 365 nm. The holistic evaluation of the numerical data corresponding different image processing channels (blue, grey, red, green) was performed by employing appropriate multivariate methods: hierarchical cluster analysis (HCA), principal component analysis (PCA), fuzzy principal component analysis (FPCA) and linear discriminant analysis (LDA) applied to the first relevant principal components. The results obtained by applying LDA method indicate a highly accurate separation of the medicinal herbs within the four groups, in good agreement with therapeutic effects and usage. According to this classification, the best image processing channels were identified for each of the investigated HPTLC plates: blue channel for HPTLC Silica gel 60 F254 (with 92.9% percent of discrimination in case of PCA and FPCA) and respectively red channel for HPTLC Silica gel 60 (with 93.9% percent of discrimination in case of FPCA). The 2D and 3D score scatterplots illustrate also the accurate and reliable discrimination between the four distinct groups.

Lipophilicity of compound is well known as vital physicochemical property of a molecule, which determines its biological activity. Nonetheless, the assessment of a lipophilicity is still problematic and focuses attention of scientists. Although, the shake-flask method is still considered as a gold standard for experimental determination of lipophilicity, currently the chromatographic approach is mostly used. Among chromatographic methods used for lipophilicity assessment, thin layer chromatography (TLC) is still one of the most popular tools. The main goal of this study was to compare classical reversed-phase thin layer chromatography (RP-TLC) and micellar TLC as potential tools for lipophilicity assessment. Micellar liquid chromatography has significantly lover environment impact than classical reversed-phase liquid chromatography. Additionally comparison of cationic and anionic surfactants (CTAB and SDS), which have different chemical properties, as modifiers of mobile phase in micellar TLC were investigated. The Quantitative Structure-Retention Relationships (QSRR) approach was used in order to present molecular mechanisms of retention in investigated chromatographic systems. The study was based on chemically diverse model set compounds, with a proved therapeutic or toxic potential. According to obtained results the micellar TLC with CTAB as surfactant can be recommended to logP prediction. The obtained QSRR models indicated that adsorption of CTAB monomers on CN modified surface of silica gel and the silanol-quaternary ammonium interactions are possible. Consequently, the reduction of interaction between molecules and free silanol, contributes to the improvement of logP predictions. These result were confirmed by regression and classification methods.

A novel method based on the fluorescence excited solely on aloin by a H 3 BO 3 derivatizing procedure, allowed its rapid and selective determination among the co-occurring components in a variety of complex matrices as several Aloes dried extracts and related commercial products. HPTLC LiChrospher silica gel 60 F254S, 20 cm × 10 cm, plates with ethyl formate: CH 3 OH:H 2 O (100:14.5:10, v/v) as the mobile phase were used. Densitometric determinations were performed in fluorescence mode, exciting wavelength 365 nm, optical filter K540 after derivatization with H 3 BO 3 . The method was validated giving rise to a dependable and high throughput procedure well suited to routine application. Aloin was quantified in the range of 110–330 ng with RSD of repeatability and intermediate precision not exceeding 2.3% and accuracy inside the acceptance limits.

The lipophilicity of a compound is a fundamental property related to pharmaceutical and biomedical activity. As many approaches are mixed together in every-day published studies, the subject needs some standardization. The paper presents a comparative study on several approaches of TLC lipophilicity determination: a single TLC run, extrapolation of a retention, principal component analysis of a retention matrix, PARAFAC on a three-way array and a PLS regression. All techniques were applied to 35 model solutes with simple molecules, using nine concentrations of six modifiers: acetonitrile, acetone, dioxane, propan-2-ol, methanol and tetrahydrofurane. The elaborated comparative analysis formed several general recommendations. Methanol and dioxane were the best modifiers, while acetonitrile gave the worst and inacceptable correlation of retention with lipophilicity. Surprisingly, good correlations were obtained for the single TLC runs and this method is underestimated in the literature. The advanced chemometric processing proposed recently, such as PCA, PARAFAC and PLS did not show a visible advantage comparing to classical methods. A need to use a robust regression and robust correlation measures, due to presence of significant outliers, was also noticed and studied.

The aim of this study was to present the usefulness of enzyme-linked immunosorbent assay (ELISA) in nicotine metabolites determination in urine and serum samples from active smokers and compare it with the reference to thin layer chromatography (TLC) with densitometry. The specific anti-cotinine antibodies were obtained from rabbit sera after sequential immunization with 4'-carboxycotinine-hemocyanine conjugate. The immunoaffinity chromatography technique with the use of self-prepared cotinine-aninohexyl-sepharose bed enabled the isolation of the specific anti-nicotine metabolites antibodies from the antiserum. Affinity of isolated antibodies to cotinine was passively immobilised on ELISA plates and competition between nicotine metabolites in samples and tracer (horseradish peroxidase-cotinine conjugate) was applied. After the washing stage the enzymatic activity of solid-phase-bound peroxidase was determined. For calibration cotinine perchlorate solutions in appropriate matrix were used. Determination ranges for serum and urine samples were from 3 to 1500 and from 3 to 5000 ng/mL, respectively. Precision within-run and between-run was below 8.7 and 11.3%; mean recovery of cotinine was 100.59% from serum and 88.56% from urine samples. The ELISA method, used in determination of the main nicotine metabolites showed high accuracy and sensitivity. However, this method was less specific than the reference technique (TLC). The high correlation coefficients, r>0.9, between the results of determined nicotine metabolites in urine by means of ELISA and TLC with densitometry confirmed the possibility of the application of ELISA method to practical monitoring of tobacco smoke exposure in large population groups.

Poloxamers (PXMs) are amphiphilic non-ionic block polymers commonly used in the cosmetic and pharmaceutical industries. In spite of the wide use of PXMs, few studies have dealt with the analysis of these polymers in pharmaceutical preparations. In this work, high-performance thin-layer chromatography (HPTLC) has been used to quantify both PXM-188 and PXM-407 in pharmaceutical preparations. The separation of these compounds was carried out using reverse phase HPTLC plates with a chloroform-methanol mixture as the mobile phase. Detection was performed densitometrically using the Dragendorff's reagent for the visualization of PXMs. Quality parameters were established, and the detection limits ranged from 24 to 47ng/spot. A good precision (day to day and run to run), with relative standard deviations

Morroniside, the most abundant iridoid glycoside of traditional Chinese medicines Fructus Corni, was shown to prevent diabetic angiopathies. During the course of our studies on its metabolism by intestinal bacteria, two metabolites (mor-1 and mor-2) were isolated and purified by thin layer chromatography (TLC) and preparative high performance liquid chromatography (HPLC), and then identified as nitrogen-containing compounds along with the known aglycones on the basis of mass spectrometry (MS), and by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Mor-1 and mor-2 were proved to be new compounds. The structures of the metabolites of morroniside detected in rat urine, bile, feces and contents of intestine after oral administration of morroniside proved to be identical with those of the microbial metabolites mor-1 and mor-2.

This paper describes an improved quality assessment method for Rhizoma et Radix Notopterygii (the rhizome and root of Notopterygium incisum Ting ex H.T. Chang or Notopterygium forbesii Boiss). The method was established by using fingerprinting and quantitation of marker compounds (isoimperatorin, notopterol and bergapten) in this herbal medicine. The authentication of Rhizoma et Radix Notopterygii using high performance thin-layer chromatography (HPTLC) fingerprinting was achieved by comparing the colors and Rf values of the bands in TLC fingerprints with those of the marker compounds. The HPLC fingerprints of 16 batches of herbal samples from different regions of China showed similar chromatographic patterns. Five peaks were selected as characteristic peaks, and three of these were identified by using LC-MS-MS techniques. The relative retention times of these characteristic peaks in the HPLC fingerprint were established as an important parameter for identification of Rhizoma et Radix Notopterygii. Finally, the pharmacologically active marker compounds isoimperatorin, notopterol and bergapten in this herb were quantitatively determined using a validated reverse-phase HPLC method.

The effect of ionizing radiation in doses between 20 and 200 kGy on physicochemical properties of fluconazole (alpha-(2,4-diflurophenyl)-alpha-(1H-triazol-1-methyl)-1H-1,2,4-triazole-1-ethanol) in the solid state was examined. A number of qualitative and quantitative methods such as scanning electron microscopy (SEM), nuclear magnetic resonance (NMR), ultraviolet (UV) and infrared (IR) spectroscopy, thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) and organoleptic analysis were used to determine and analyse any changes resulting from irradiation. A change in colour from white to cream was observed at even smallest dose (20 kGy) and as the dose increased the colour deepened from salmon pink to orange at the highest dose of 200 kGy. The UV method showed an increase in absorbance at lambda(max) and an appearance of an additional band in the range 280-310 nm for irradiated samples. These changes were associated with the appearance of one to two decomposition products observed by TLC. Depending on the dose of radiation, the HPLC method detected between 2 and 3 radiolysis products and the decreasing fluconazole content from 0.48 to 7.12%. The remaining analytical methods (SEM, IR and NMR) did not provide any conclusive information in respect of radiological stability of fluconazole. The results indicate that fluconazole is a compound of low radiological stability and should not be sterilized using gamma, beta or E-beam radiation.

A simple, sensitive, precise and rapid high-performance thin-layer chromatographic (HPTLC) method of analysis for artemether both as a bulk drug and in pharmaceutical formulations was developed and validated. The method employed TLC aluminum plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-ethyl acetate-formic acid (8:2:0.3, v/v/v) as mobile phase. Densitometric analysis of artemether was carried out in the reflectance mode at 565 nm. The system was found to give compact spots for artemether (R(f) value of 0.50+/-0.03). The linear regression analysis data for the calibration plots showed good linear relationship with r(2)=0.9904 in the concentration range 200-1000 ng per spot. The mean value of correlation coefficient, slope and intercept were 0.9904+/-0.011, 7.27+/-0.11 and 166.24+/-56.92, respectively. The method was validated for precision, accuracy, recovery and robustness. The limits of detection and quantitation were 65.91 and 197.74 ng per spot, respectively. The method has been successfully applied in the analysis of lipid based parenteral formulations and marketed oral solid dosage formulation.

1,3-dimethylbutylamine (DMBA), is a CNS stimulant, which has recently been identified in multiple dietary supplements and sometimes labeled as a natural constituent of Pouchung tea. DMBA is an homologue of 1,3-dimethylamylamine (DMAA) which the US Food and Drug Administration has attempted to remove from all dietary supplements after DMAA consumption was linked to strokes, heart disease, and sudden death. To address questions concerning the natural origin of DMBA, three independent analytical methods were developed for analyzing authentic tea samples and dietary supplements. A high performance thin layer chromatography (HPTLC) method was developed for the fast screening and chemical fingerprint analysis. Chiral Gas Chromatography-Mass Spectrometry (GC-MS) was used to determine the enantiopurity and a validated Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry (UHPLC-QToF-MS) method was developed for the quantification of DMBA. Using these techniques the presence of DMBA was confirmed using a reference standard and was not detected in any of 25 authentic or commercial samples of Camellia sinensis tea leaves (green tea, black tea, Oolong tea, and Pouchung tea). Of 13 dietary supplements tested, 11 contained DMBA in racemic form and ranged from 0.1 to 214mg per daily dose.

The chromatographic indices log kwIAM have been determined by HPLC on an immobilized artificial membrane column for a set of coumarin derivatives. The investigated compounds contain substituted amidoximes, or substituted heterocycles directly attached to the coumarinic skeleton. The log kwIAM values were compared to previously reported data of octanol–water partition coefficients and extrapolated capacity factors determined by reversed phase HPLC and TLC. The log kwIAM values of the investigated compounds were found to be comparable with the corresponding log P values, although they constitute a lower lipophilicity scale due to the reduced hydrophobic environment of the IAM stationary phase. These features were exemplified in their interrelationship with a slope close to unity and a large negative intercept. In contrast their comparison with the corresponding HPLC and RP-TLC capacity factors revealed differences in the retention mechanism reflected in slopes lower than unity, which were postulated to be due to secondary interactions under the reversed phase chromatographic conditions. However, conformational effects in the molecular structures of the coumarin derivatives were found to have a similar impact in their affinity for the IAM and octadecyl silane stationary phases, while they did not affect their octanol–water partitioning.

Stability-indicating high performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and first-derivative of ratio spectra (1DD) methods are developed for the determination of piretanide in presence of its alkaline induced degradates. HPLC method depends on separation of piretanide from its degradates on mu-Bondapak C18 column using methanol:water:acetic acid (70:30:1, v/v/v) as a mobile phase at flow rate 1.0 ml/min and UV detector at 275 nm. TLC densitometic method is based on the difference in Rf-values between the intact drug and its degradates on thin-layer silica gel. Iso-propanol:ammonia 33% (8:2, v/v) was used as a developing mobile phase and the chromatogram was scanned at 275 nm. The derivative of ratio spectra method (1DD) depends on the measurement of the absorbance at 288 nm in the first-derivative of ratio spectra for the determination of the cited drug in the presence of its degradates. Calibration graphs of the three suggested methods are linear in the concentration ranges 0.02-0.3 microg/20 microl, 0.5-10 microg/spot and 5-50 microg/ml, with mean percentage recovery 99.27+/-0.52, 99,17+/-1.01 and 99.65+/-1.01%, respectively. The three proposed methods were successfully applied for the determination of piretanide in bulk powder, laboratory-prepared mixtures and pharmaceutical dosage form with good accuracy and precision. The results were statistically analyzed and compared with those obtained by the official method. Validation of the method was determined with favourable specificity, linearity, precision, and accuracy was assessed by applying the standard addition technique.

Colchicine, (S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo-(a(-heptalen-7-yl)-acetamide, is the main alkaloid contained in Colchicum autumnale (meadow saffron). There are known colorimetric, spectrophotometric, volumetric, potentiometric, voltametric, gravimetric and various chromatographic methods for quantitative determination of colchicine, each of them presenting a series of advantages and disadvantages. As an alternative, we proposed the use of a densitometric determination for colchicine allowing the determination of this alkaloid from pharmaceutical products, as well from seeds of meadow saffron. The total alkaloid extract was separated by Thin-Layer Chromatography using Silicagel 60F254 layers and a mixture of chloroform:acetone:diethylamine (5:4:1) as mobile phase. The same conditions were used for the determination from pharmaceutical products. Densitometric measurements were carried out at the absorption maximum (350 nm) of colchicine, the determinations being made by reflectance and by fluorescence. The peaks were optimized regarding to their area and shape by varying four scanning parameters (slit width and height, number of measurements and scanning speed). We established the calibration plot using pure colchicine in the range 50–600 ng mL−1. The proposed method could be widely used in the pharmaceutical industry for the quick and accurate quantitative determination of colchicine because it eliminates the interferences given by other bioactive or degradation compounds. The method was characterized by validation parameters (linearity, accuracy, fidelity, sensitivity) and it was established its performances in comparison with an HPLC method and an official quantitative determination from the Romanian Pharmacopoeia X edition respectively.

A sensitive, selective, precise and robust high-performance thin-layer chromatographic method of analysis of E and Z stereoisomers of guggulsterone (the hypolipidemic agent in the gum-resin exudates of Commiphora mukul) both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene–acetone (9:1, v/v). Densitometric analysis of guggulsterone was carried out in the absorbance mode at 250 nm. This system was found to give compact spots for E- and Z-guggulsterone (Rf value of 0.38±0.02 and 0.46±0.02, respectively) following double development of chromatoplates with the same mobile phase. The linear regression analysis data for the calibration plots for E- and Z-guggulsterone showed good linear relationship with r2=0.9977±0.054 and 0.9975 ± 0.068, respectively, in the concentration range of 100–6000 ng/spot. The mean value of slope and intercept were 0.11±0.006 and 0.11±0.005, 14.26±0.56 and 10.92±0.76, respectively, for E- and Z-guggulsterone. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 12, 10 and 24, 20 ng/spot, respectively, for E- and Z-guggulsterone. Statistical analysis proves that the method is repeatable and selective for the estimation of the said drug. Since the proposed mobile phase effectively resolves the E- and Z-isomers of guggulsterone, this HPTLC method can be applied for identification and quantitation of these isomers in herbal extracts and pharmaceutical dosage form.

Surfaces of lungs are covered by the surfactant, an aqueous mixture of different phospholipids (PL) and proteins. Although the surfactant represents a relatively simple mixture of only a few PL (primarily phosphatidylcholine (PC) and phosphatidylglycerol (PG)), reliable methods of routine lipid analysis of the surfactant are still lacking. It will be shown that matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS) represents a suitable technique for the differentiation of the apolar components of the surfactant of different species. Samples of man and minipig are used in this study since both are known to vary in their PL composition. PL of surfactant were separated by thin-layer chromatography (TLC) and the obtained subfractions subjected to MALDI-TOF MS analysis in order to monitor the presence of even minor PL species. It will be shown that besides PG and PC, also phosphatidylethanolamine, -inositol and sphingomyelin can be detected in surfactant of man, whereas only sphingomyelin could be detected in the minipig sample.

Four stability-indicating methods were developed for the determination of sumatriptan succinate in the presence of its degradation products. The first method depends on the quantitative densitometric evaluation of thin-layer chromatography of sumatriptan succinate in the presence of its degradation products without any interference. Cyclohexane-dichloromethane-diethylamine (50:40:10 v/v/v) was used as a mobile phase and the chromatogram was scanned at 228 nm. This method determines sumatriptan succinate in the concentration range l-8 microg per spot with mean percentage recovery 100.52+/-1.23%. The second and third methods depend on the use of first-derivative (D(1)) and second-derivative (D(2)) spectrophotometry at 234 and 238 nm, respectively. These methods determine the drug in the concentration range 1.25-10 microg x ml(-1) with mean percentage recovery 99.91+/-1.01% and 99.96+/-1.13% for (D(1)) and (D(2)), respectively. The fourth method depends on the use of ratio derivative spectrophotometric technique. The amplitude in the first derivative of the ratio spectra at 235 nm was selected to determine the cited drug in the presence of its degradation products. Calibration graph is linear in the concentration range 1.25-10 microg x ml(-1) with mean percentage recovery 100.19+/-1.19%. The suggested methods were successfully applied for determining sumatriptan succinate in bulk powder, laboratory-prepared mixtures and pharmaceutical dosage forms (Imigran tablet) with good accuracy and precision. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by the reported method.

The enantiomeric resolution of certain 2-arylpropionic acids was achieved on thin silica gel plates impregnated with optically pure L-(-)-serine as chiral selector. The mobile phase enabling successful resolution of (+/-)-ibuproxam and (+/-)-ketoprofen was acetonitrile-methanol-water (16:4:0.5, v/v/v) and (16:3:0.5, v/v/v) for (+/-)-tiaprofenic acid. The spots were detected with iodine vapors and the detection limits were found to be different for each of the 2-arylpropionic acid, ranging between 0.25 and 0.50 microg/ml. The effect of concentration of the impregnating chiral selector, temperature and pH on resolution has been studied. The procedure was applied successfully to resolve commercial ampoules of ketoprofen dosage formulation.

The UV irradiated aqueous solutions of folic acid at pH 2-10 degrade to give pterin-6-carboxylic acid and p-aminobenzoyl-L-glutamic acid under aerobic conditions. These photoproducts have been identified by TLC, HPLC and Spectrophotometric techniques. A reaction scheme for the photodegradation pathways of folic acid leading to the formation of the photoproducts in acid and alkaline media has been proposed which involves the participation of an enamine intermediate.

The British Pharmacopoeia (BP) monograph for cyproheptadine HCl tablets requires a ‘Related substances’ thin-layer chromatography (TLC) test. This test revealed an extraneous spot with an R f of 0.1 in certain cyproheptadine HCl tablets that were under ambient retention conditions as well as those on stability programs. An investigation utilizing LC-MS, direct infusion MS, NMR, and organic synthesis has identified that the spot results from the N -oxide of cyproheptadine (a genuine degradate) and a co-eluting cyproheptadine–dichloromethane adduct, an artifact formed during the sample extraction step in which dichloromethane is used in the extracting solvent.

Three methods are presented for the determination of Nicergoline in presence of its hydrolysis-induced degradation product. The first method was based on measurement of the first derivative of ratio spectra amplitude of Nicergoline at 291 nm. The second method was based on separation of Nicergoline from its degradation product followed by densitometric measurement of the spots at 287 nm. The separation was carried out on HPTLC silica gel F(254) plates, using methanol-ethyl acetate-glacial acetic acid (5:7:3, v/v/v) as mobile phase. The third method was based on high performance liquid chromatographic (HPLC) separation and determination of Nicergoline from its degradation product on a reversed phase, nucloesil C(18) column using a mobile phase of methanol-water-glacial acetic acid (80:20:0.1, v/v/v) with UV detection at 280 nm. Chlorpromazine hydrochloride was used as internal standard. Laboratory prepared mixtures containing different percentages of the degradation product were analysed by the proposed methods and satisfactory results were obtained. These methods have been successfully applied to the analysis of Nicergoline in Sermion tablets. The validities of these methods were ascertained by applying standard addition technique, the mean percentage recovery +/- R.S.D.% was found to be 99.47 +/- 0.752, 100.01 +/- 0.940, 99.75 +/- 0.740 for the first derivative of ratio spectra method, the HPTLC method and the HPLC method, respectively. The proposed methods were statistically compared with the manufacturer's HPLC method of analysis of Nicergoline and no significant difference was found with respect to both precision and accuracy. They have the advantage of being stability indicating. Therefore, they can be used for routine analysis of the drug in quality control laboratories.

Five new selective, precise and accurate methods are described for the determination of clozapine in the presence of its main degradation product. Method A utilizes the second and third derivative spectrophotometry at 315 and 305 nm, respectively. Method B is RSD(1) spectrophotometric method based on the simultaneous use of the first derivative of ratio spectra and measurement at 295 nm. Method C is a pH-induced difference (delta A) spectrophotometry using UV measurement at 325 nm. Method D is a densitometric one, after separation on silica gel plate using methanol: water as mobile phase, and the spot was scanned at 295 nm. Method E is RP-HPLC using acetonitrile: water (40:60 v/v) as mobile phase at a flow rate of 1 ml/min and UV detection was at 295 nm. Regression analysis showed good correlation in the concentration ranges 3-10, 4-10, 10-25 micro g/ml, 200-1000 ng/spot, 5-100 micro g/ml with percentage recoveries of 99.4+/-0.28, 99.8+/-0.20, 100.05+/-0.11, 99.41+/-0.34, 100.11+/-0.07 and 100.07+/-0.05% for methods A, B, C, D and E, respectively. These methods are suitable as stability indicating methods for the determination of clozapine in the presence of its main degradation product either in bulk powder or in pharmaceutical formulations.

A binary mixture of terbinafine hydrochloride and triamcinolone acetonide was determined by three different methods. The first one concerned with determination of both drugs using first derivative (D(1)) spectrophotometric technique at 297 and 274 nm over concentration ranges of 5-30 and 4-24 microg ml(-1), with mean percentage accuracies 99.90+/-0.67 and 100.25+/-0.49, respectively. The second method depends on ratio-spectra 1st derivative (RSD(1)) spectrophotometry at 298 and 248 nm over the same concentration ranges with mean percentage accuracies 100.22+/-0.51 and 99.93+/-0.56, respectively. The spectrodensitometric analysis provides a rapid and precise method for the separation and quantitation of both terbinafine hydrochloride and triamcinolone acetonide. The method depends on the quantitative densitometric evaluation of thin layer chromatogram of terbinafine hydrochloride and triamcinolone acetonide at 283 and 238 nm over concentration ranges of 5-25 and 2.5-22.5 microg spot(-1), with mean percentage accuracies 100.66+/-0.51 and 100.27+/-0.73, respectively. The suggested procedures were checked using laboratory prepared mixtures and were successfully applied for the analysis of their pharmaceutical preparations. The three methods retained their accuracy and precision when applying the standard addition technique. The results obtained by applying the proposed methods were statistically analysed and compared with those obtained by a reported method.

In high performance thin layer chromatography some form of optimization is necessary if complete separation of all components is required. The selection of mobile phase composition is one of the most important components of an optimization strategies. The aim of this paper is the separation of the N-alkyl phenothiazine sulfones by high performance thin layer chromatography using an optimum mobile phase system. The optimum composition of mobile phase (toluene–ethyl ether–chloroform, 30:50:20, v/v) are given by the maximum of objective function (Fobj=10.6110).

A chromatographic-densitometric method for identification and quantitation of diclofenac and its impurities, i.e. 1-(2,6-dichlorophenyl)indolin-2-one and indolin-2-one in pharmaceutical preparations and model solutions was developed. The effect of pH, temperature and ultra violet (UV) radiation on diclofenac's concentration was investigated. Chromatographic separation was performed on TLC silica gel coated plates with the mobile phase: cyclohexane-chloroform-methanol (12:6:1, v/v/v). Densitometric detection was carried out in UV at lambda=248 nm. The conditions for good separation and the detection limit were established. The recovery for diclofenac was 99.20%, for 1-(2,6-dichlorophenyl)indolin-2-one--92.34% and for indolin-2-one--95.85%. The method was used for quality assessment of diclofenac in pharmaceutical preparations. Reliable results comparable to those determined by high performance liquid chromatography (HPLC) were obtained.

Two stability-indicating methods were developed for the determination of doxazosin mesylate ( I ) and celecoxib ( II ) in the presence of their degradation products. The first method depends on the use of first derivative spectrophotometry ( D 1 ) at 256, 269 nm for ( I ) and ( II ), respectively. This method determines ( I ) and ( II ) in concentration ranges of 0.8–12 and 1–20 μg ml −1 with mean percentage accuracies of 99.21±0.88 and 99.59±1.67% for ( I ) and ( II ), respectively. The second method depends on the quantitative densitometric evaluation of thin-layer chromatography of ( I ) and ( II ) in the presence of their degradation products without any interference. Methylisobutyl ketone–glacial acetic acid–water (20:10:10) was used as a mobile phase for ( I ) and cyclohexane–dichloromethane–diethyleamine (50:40:10) for ( II ). The chromatograms were scanned at 248 and 253 nm for ( I ) and ( II ), respectively. This method determines ( I ) and ( II ) in concentration ranges of l–4 μg per spot for both drugs with mean percentage accuracies of 100.19±0.95 and 99.91±1.95% for ( I ) and ( II ), respectively. The suggested methods were used to determine doxazosin mesylate and celecoxib in bulk powder, laboratory-prepared mixtures and pharmaceutical dosage forms (cardura tablet and celebrex capsule). The results obtained by applying the proposed methods were statistically analysed and compared with those obtained by the reported methods.

Mangifera indica, commonly called mango or amra belonging to a family of Anacardiaceae, is an important medicinal plant widely used in a variety of Ayurvedic preparations. Extract of its bark, leaves, flowers and kernels are being extensively used for curing various chronic diseases. Mango wood is used in yagya as base fire through which medicated smoke is generated. Three new compounds have been isolated from methanolic and hexane extracts of stem bark: 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl)ester and 9,12-tetradecadiene-1-ol-acetate from the hexane extract and 3-chloro-N-(2-phenylethyl) propanamide from the methanolic extract. These were first separated by thin layer chromatography and later in a silica gel column and identified by characteristic infrared bands corresponding to respective functional groups. The compounds were further confirmed on the basis of GC-MS fragmentation pattern after comparing the data with NIST mass spectral database. All three compounds exhibited antimicrobial activity due to triterpenoids and flavonoids. Elemental analyses by INAA show it to be enriched in essential nutrient elements such as Ca, Fe, K, Mn and Zn which all play an important role in enzymatic processes.

This research presents the results obtained from analysis by thin layer chromatography (TLC) of some free amino acids from sanguine plasma samples in the different degree progress in maladies: diabetes, renal syndrome and hepatic cirrhosis. The chromatograms were evaluated with a Shimadzu CS-9000 dual-wavelength flying-spot scanner. Better results were obtained in the case of hepatic cirrhosis.

A high-performance thin-layer chromatographic method (HPTLC) has been developed for the determination and the purity control of a synthetic fluoroquinolone antibiotic ciprofloxacin hydrochloride in coated tablets when desfluoro compound, ethylene diamine compound, by-compound A and fluoroquinolonic acid are considered as impurities. Silica gel F254 was used as a stationary phase and a mixture of acetonitrile, ammonia 25%, methanol and methylene chloride (1:2:4:4, v/v/v/v) as a mobile phase. The method was validated in terms of linearity (range), selectivity (placebo and related compounds), precision, accuracy (Recovery), system suitability (repeatability, peak symmetry, resolution) and impurities limit of detection (LOD). The analysis of variance (ANOVA) and t-test were applied to correlate the results of ciprofloxacin hydrochloride determination in coated tablets by means of the HPTLC method and the official British Pharmacopoeia (BP 1999) high-performance liquid chromatographic (HPLC) method.

Different spectrophotometric and HPTLC-densitometric methods are presented for the simultaneous determination of lisinopril and hydrochlorothiazide in pharmaceutical tablets. The spectrophotometric methods include third derivative (3D) ultraviolet spectrophotometry with zero crossing measurement at 217.4 and 233.4 nm, second derivative of the ratio spectra with measurement at 214.3 and 228.0 nm; both classical least squares and principal component regression were applied to the UV absorption and first derivative spectra of the mixture. The HPTLC method was based on separation of both drugs followed by densitometric measurements of their spots at 210 and 275 nm for lisinopril and hydrochlorothiazide, respectively. The separation was carried out on Merck HPTLC aluminum plates of silica gel 60 F254, using chloroform–ethylacetate–acetic acid (10:3:2 by vol.) as mobile phase. The linear and second order polynomial were used for the regression equation of lisinopril and hydrochlorothiazide, respectively.

The combination of pseudoephedrine and cetirizine is widely used in the treatment of allergic rhinitis. A rapid, selective and stability indicating high performance thin layer chromatographic method was developed and validated for their simultaneous estimation in pharmaceutical dosage forms. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of ethyl acetate-methanol-ammonia (7:1.5:1, v/v/v). This system was found to give compact spots for both pseudoephedrine (Rf value of 0.69+/-0.01) and cetirizine (Rf value of 0.38+/-0.01). Also the degraded products were well separated from the pure drugs. Spectrodensitometric scanning-integration was performed at a wavelength of 240 nm. The polynomial regression data for the calibration plots showed good linear relationship with r(2)=0.9947 in the concentration range of 10-26 microg for pseudeophedrine and 200-1200 ng for cetirizine with r(2)=0.9973. The method was validated for precision, accuracy, ruggedness and recovery. The minimum detectable amounts were found to be 2 microg and 500 pg for pseudoephedrine and cetirizine, respectively. The limits of quantitation were found to be 6 microg for pseudoephedrine and 800 pg for cetirizine. Both the drugs do not undergo degradation under acidic and basic conditions. The samples degraded with hydrogen peroxide showed additional peaks at Rf values of 0.75 and 0.28 for pseudoephedrine and cetirizine, respectively. This indicates that both the drugs are susceptible to oxidation. Statistical analysis proves that the method is reproducible and selective for the simultaneous estimation of pseudoephedrine and cetirizine. As the method could effectively separate the drugs from their degradation products, it can be employed as a stability indicating one.

A thin layer chromatographic-densitometric method for identification and quantitation of neomycin sulfate, polymixin B sulfate, zinc bacytracin and auxiliary substances (methyl and propyl hydroxybenzoates) in ophthalmic ointment was developed. To separate these constituents the silica gel coated TLC plates and two mobile phases were used. The suitable mobile phases were: methanol-n-butanol-ammonia 25%-chloroform (14:4:9:12, v/v/v/v) for determination of antibiotics and n-pentane-glacial acetic acid (66:9, v/v) for methyl and propyl hydroxybenzoates. The antibiotic chromatograms were detected by using ninhydrin ethanol solution, while densitometric measurements were made at lambda = 550 nm. Hydroxybenzoates were identified by UV measurements at lambda = 260 nm. The constituents under consideration were well separated at sufficient detection level. The recovery for all constituents ranged from 98.08% to 104.95%.

Timolol was the first beta blocker to be used as an anti-glaucoma agent and to date remains as the standard because none of the newer beta blockers were found to be more effective. The high performance thin layer chromatographic method of analysis of timolol maleate is reported. The mobile phase selected was ethyl acetate-methanol-isopropyl alcohol-ammonia (25%) (80:20:2:1, v/v/v/v). The calibration curve of the drug was linear in the range of 100-600 ng. The spectrodensitometric analysis was carried out at 294 nm. The mean (+/- RSD) values of slope, correlation coefficient and intercept were 2487.5 (+/- 0.9), 0.996 (+/- 0.081) and 90463 (+/- 1.1), respectively. The system precision and the method precision were excellent with an RSD of 2.8 and 1.004, respectively. The limits of detection and quantitation were 10 and 40 ng, respectively. The mean percent recovery was found to be 98.6. Timolol maleate was degraded by exposing the drug to heat, acid and base. The degraded products were found to be well separated from the pure drug with significantly different Rf values suggesting a stability indicating analysis method for quantification of timolol maleate in pharmaceutical preparations and as bulk drug. The method was utilized to analyze timolol maleate from conventional eye drops and novel sustained release solid polymeric ocular inserts and oral preparations. The reported method is simple, selective, precise, accurate, time saving and economic as compared to reported HPLC methods.