Current Pharmaceutical Analysis - Volume 17, Issue 1, 2021

To develop effective and safe drugs and to take them to the market in short period of time is the mission of pharmaceutical research companies. A selection of few of the lead compounds are done for the evaluation of safety and their ADMET (absorption, distribution, metabolism, excretion and toxicology) properties are tested in in-vitro (test systems), in-vivo (living organisms) and in-silico (computational methods). From initial stages to final stages of modern drug discovery processes, the vital tool for detecting and characterizing metabolites is MS (Mass spectrometry) hyphenated with other techniques. The methods used for generation of metabolites are in vitro techniques and cell lines (containing expressing drug metabolizing enzymes and heterologous genes). The use of HPLC-MS/UPLC-MS and high resolution MS, enables the in depth metabolite detection and profiling studies and it may also be likely to identify and characterize the site and types of biotransformation.

The hyphenation of Ultra-Performance Liquid performance (UPLC) with mass spectrometry (MS) has emerged as a powerful tool in analytical research due to its advanced sensitivity, resolution and speed. Its advanced instrumentation, specialized columns, separation at ultra-high pressure and sophisticated software are the distinguishing features as compared to the traditional separating techniques. It has a wide range of applications in various fields such as analysis of food stuffs, drug metabolites, beverages, toxicology, soil samples and micronutrient analysis. In the present compilation, authors have highlighted the applicability of UPLC-MS in the analysis of food stuffs and drug metabolites along with the various optimized analytical conditions and obtained results.

Background: Tibolone is a synthetic steroid commercialized by Organon under the brand name Livial (Org OD14), which is used in hormone therapy for menopause management and treatment of postmenopausal osteoporosis. Tibolone is defined as a selective tissue estrogenic activity regulator (STEAR) demonstrating tissue-specific effects on several organs such as brain, breast, urogenital tract, endometrium, bone and cardiovascular system. Aims: This work aims to (1) present an overview of important published literature on existing methods for the analysis of tibolone and/or its metabolites in pharmaceutical formulations and biological fluids and (2) to conduct a critical comparison of the analytical methods used in doping control, pharmacokinetics and pharmaceutical formulations analysis of tibolone and its metabolites. Results and Conclusion: The major analytical method described for the analysis of tibolone in pharmaceutical formulations is High Pressure Liquid Chromatography (HPLC) coupled with ultraviolet (UV) detection, while Liquid Chromatography (LC) or Gas Chromatography (GC) used in combination with Mass Spectrometry (MS) or tandem mass spectrometry (MS/MS) is employed for the analysis of tibolone and/or its metabolites in biological fluids.

Background: The Glassy Carbon Electrode (GCE) was modified with zinc oxide nanoparticles to enhance the electrocatalytic activity of the redox behavior of cefotaxime ion. ATOMIC Force Microscopy (AFM) photographic studies showed the nanorod like structure of the zinc oxide, which was coated uniformly on the electrode surface. Methods: The zinc oxide nanorod modified electrode was used as novel voltammetric determination of cefotaxime. The results of voltammetric behavior are satisfactory in the electro oxidation of cefotaxime, and exhibit considerable improvement compared to glassy carbon electrode. Results: Under the optimized experimental conditions, the ZnO nanorod modified electrode exhibit better linear dynamic range from 300 ppb to 700 ppb with lower limit of detection 200 ppb for the stripping voltammetric determination of cefotaxime. Conclusion: The pharmaceutical and clinical formulation of cefotaxime was successfully applied for accurate determination of trace amounts on ZnO nanomateials modified electrode.

Background: Zishen Tongguan (ZSTG) capsules were prepared at the Affiliated Hospital of Nanjing University of Chinese Medicine and have been proven to be clinically effective for treating pyelonephritis and benign prostatic hyperplasia. However, the quality standards are not ideal; a comprehensive study of the “quality markers” (Q-markers), the chemicals inherent in traditional Chinese medicine and its preparations, has not been carried out. Experimental Methods: In this paper, a sensitive and specific ultra-high-performance liquid chromatographictandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of eight potential Q-markers of ZSTG, including timosaponin A3, berberine, jatrorrhizine, phellodendrine, palmatine, mangiferin, neomangiferin, and timosaponin BII. A Kromasil 100-3.5 C18 column was used with a mobile phase of 0.2% formic acid with acetonitrile, and gradient elution at a flow rate of 0.2 mL/min was achieved in 13 minutes and used for separation. Detection was performed in positive/negative mode with multiple reaction monitoring (MRM). Results: The analytical method was validated in terms of the sensitivity, linearity, accuracy, precision, repeatability, stability and recovery. The method established here was successfully applied to study the potential Q-markers in 8 batches of commercial samples, which demonstrated its use in improving the quality control of ZSTG. Conclusion: The developed method had high repeatability and accuracy and was suitable for the simultaneous analysis of multiple Q-markers, which may provide a new basis for the comprehensive assessment and overall quality control of ZSTG.

Background: Sample preparation is crucially important for the capillary electrophoretic measurement of the bioactive constituents in Citri Reticulatae Pericarpium because conventional solvent extraction is time-consuming and the solvent peaks seriously interfere with the measured capillary electropherograms. Objective: The objective of the present study is to establish far infrared-assisted sample preparation approaches for the analysis of Citri Reticulatae Pericarpium. Methods: Synephrine and hesperidin in Citri Reticulatae Pericarpium were determined by capillary electrophoresis in combination with far infrared-assisted sample extraction and solvent removal. Results: The effects of detection potentials, irradiation times and the voltages applied to the infrared generator were investigated to acquire the optimal assay conditions. Synephrine and hesperidin could be well separated within 6 min at a separation voltage of 9 kV in an alkaline borate solution. Satisfactory linearity was observed over the concentration range of 0.001 to 1 mM with the detection limits of 0.43 and 0.52 μM for synephrine and hesperidin, respectively. The results exhibited that far infrared irradiations could enhance the efficiencies of sample extraction and solvent removal during the sample preparation of Citri Reticulatae Pericarpium. The extraction time was significantly reduced to 6 min while the interference of the solvent peaks towards the electropherograms was eliminated. Conclusion: Far infrared-accelerated extraction and solvent removal were employed in the capillary electrophoretic determination of the bioactive constituents in Citri Reticulatae Pericarpium with satisfactory results. The ease, simplicity, efficiency and low cost of the novel sample preparation approaches indicate they may find a wide range of applications.

Background: Zaoren Anshen Prescription (ZAP) is widely used as a classic Chinese Traditional Medicine (TCM) prescription for the treatment of palpitations and insomnia in China. Some studies have identified the main active components for its anti-insomnia effect and observed changes of some endogenous components that are closely related to its anti-insomnia effect. However, simultaneous determination of four monoamine neurotransmitters and seven effective components of ZAP and the investigation of their distribution in tissues by using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) have not been reported. Methods: An ultra-performance liquid chromatography with tandem mass spectrometry method was developed and validated for simultaneous quantification of four monoamine neurotransmitters (norepinephrine, dopamine, 5-hydroxy tryptamine and 5-hydroxyindoleacetic acid) and seven prescription components (danshensu, protocatechualdehyde, spinosin, 6``` -feruylspinosin, salviaolic acid B, schisandrin and deoxyschisandrin) in rats’ tissues. Tissue samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and 0.01% formic acid water. An electrospray ionization triple quadrupole concatenation mass spectrometer was set to switch between positive and negative modes in single run time. All the components were quantitated by multiple-reaction monitoring scanning. Results: The lower limits of quantitation for all analytical components were 0.78 ng/mL-1.99 ng/mL in the heart, liver, spleen, lung, kidney and brain. All the calibration curves displayed good linearity (r> 0.99544). The precision was evaluated by intra-day and inter-day assays, and the relative standard deviation (RSD) values were all within 12.67%. The relative errors of the accuracy were all within ± 19.88%. The recovery ranged from 76.00% to 98.78% and the matrix effects of eleven components were found to be between 85.10% and 96.40%. Conclusion: This method was successfully applied to study the distribution of seven components from ZAP and the concentration changes of four monoamine neurotransmitters after oral ZAP in six tissues.

Background: Phenylalanine is a significant biomarker for various diseases like phenylketonuria, gastric cancers, and ischemic stroke according to recent studies. Method: In the present study; a simple, sensitive, selective and novel analytical method was validated by using an ultrafiltration-based extraction and LC-MS/MS quantification of phenylalanine in human plasma using ¹³C phenylalanine heavy isotope. Amicon® Ultra Centrifugal Filter was used for ultrafiltration. Parameters affecting LC separation and MS/MS detection were investigated and optimized. Chromatographic separation was achieved on a Merck SeQuant ZIC-HILIC (100x4.6 mm, 5 μm) at a column temperature of 40°C using a mobile phase of mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid (50:50 v/v) at a flow rate of 0.35 mL/min. The transitions m/z 167→121 for <¹³C phenylalanine, m/z 166→120 for phenylalanine itself were monitored using the MRM mode. Result: The assay was linear concentration range of 0.0025 μg/mL to 1.20 μg/mL (R²=0.999). The developed method was validated according to FDA guidelines. The method was found linear, sensitive, precise, accurate, and selective.

Introduction: Novel manipulations of the well-known classical least squares multivariate calibration model were explained in detail as a comparative analytical study in this research work. In addition to the application of plain classical least squares model, two preprocessing steps were tried, where prior to modeling with classical least squares, first derivatization and orthogonal projection to latent structures were applied to produce two novel manipulations of the classical least square-based model. Moreover, spectral residual augmented classical least squares model is included in the present comparative study. Quantitative determination of pyridostigmine bromide in the presence of its two related substances; impurity A and impurity B was considered as a case study to construct the comparison. Method: 3 factor 4 level design was implemented constructing a training set of 16 mixtures with different concentrations of the studied components. To investigate the predictive ability of the studied models; a test set consisting of 9 mixtures was constructed. Results: The key performance indicator of this comparative study was the root mean square error of prediction for the independent test set mixtures, where it was found 1.367 when classical least squares applied with no preprocessing method, 1.352 when first derivative data was implemented, 0.2100 when orthogonal projection to latent structures preprocessing method was applied and 0.2747 when spectral residual augmented classical least squares was performed. Conclusion: Coupling of classical least squares model with orthogonal projection to latent structures preprocessing method produced significant improvement of the predictive ability of it.

Background: A simple and sensitive Ultra Performance Liquid Chromatography-Mass Spectrometry method was developed and validated to measure the concentrations of Alogliptin (ALO), Linagliptin (LIN), Saxagliptin (SAX), and Sitagliptin (SIT) using Pioglitazone (PIO) as an internal standard. Methods: Chromatographic separation of six gliptins was achieved on a C-18 column (100×2.1 mm, 2.7 μm) using a mobile phase consisting of formic acid in water, 0.1%v/v: acetonitrile in gradient elution. Electrospray ionization (ESI) source was operated in the positive ion mode. Targeted MS/MS mode on a QTOF MS was used to quantify the drug utilizing the transitions of 340.1(m/z), 473.2 (m/z), 316.2 (m/z), 408.1 (m/z), and 357.1 (m/z) for ALO, LIN, SAX, SIT and PIO respectively. Results: As per ICH Q2R1 guidelines, a detailed validation of the method was carried out and the standard curves were found to be linear over the concentration ranges of 1516.0-4548.1 ng mL^-1, 519.8- 1559.4 ng mL^-1, 1531.4-4594.3 ng mL^-1and 1519.6-4558.8 ng mL^-1 for ALO, LIN, SAX and SIT respectively. Precision and accuracy results were within the acceptable limits. The mean recovery was found to be 98.8 _ 0.76 % (GEM), 102.2 _ 1.59 % (LIN), 95.3 _ 2.74 % (SAX) and 99.2 _ 1.75 % (SIT) respectively. Conclusion: The optimized validated UPLC QTOF-MS/MS method offered the advantage of shorter analytical times and higher sensitivity and selectivity. The optimized method is suitable for application in quantitative analysis of pharmaceutical dosage forms for QC laboratory.

Background: Tanshinone IIA (TIIA), protocatechuic aldehyde (PA), danshensu (DSS), salvianolic acid B (SAB) and hydroxysafflor yellow A (HSYA) are the major components of Salvia miltiorrhiza Bge. (Danshen) and Carthamus tinctorius L. (Honghua) herbal pair. These active components may contribute to the potential synergistic effects of the herbal pair. Objective: This study aimed to investigate the metabolites of TIIA, PA, DSS, SAB and HSYA in zebrafish, and to explore the influence of HSYA on the metabolism of TIIA, PA, DSS, and SAB. Method: 48 h post-fertilization zebrafish embryos were exposed either to each compound alone, TIIA (0.89 μg/mL), PA (0.41 μg/mL), DSS (0.59 μg/mL), SAB (2.15 μg/mL), and HSYA (1.83 μg/mL) and in combination with HSAY (1.83 μg/mL). The metabolites of TIIA, PA, DSS, SAB, and HSYA in zebrafish were characterized using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) and quantitatively determined by HPLC-MS with single and combined exposure. Results: Among the 26 metabolites detected and characterized from these five compounds, methylation, hydroxylation, dehydrogenation, hydrolysis, sulfation and glucuronidation were the main phase I and phase II metabolic reactions of these compounds, respectively. Furthermore, the results showed that HSYA could either enhance or reduce the amount of TIIA, PA, DSS, SAB, and their corresponding metabolites. Conclusion: The results provided a reference for the study on drug interactions in vivo. In addition, the zebrafish model which required much fewer amounts of test samples, compared to regular mammal models, had higher efficiency in predicting in vivo metabolism of compounds.

Background: Grape Seed Procyanidins (GSP) refers to a type of natural polyphenols that have to roust antioxidant capacity. Studies have shed light on the fact that GSP significantly impacts the alleviation of Alzheimer's Disease (AD). Objective: This study aimed at investigating whether there exists a pharmacokinetics difference in GSP between normal and AD rats, a rapid UPLC-MS/MS methodology, for the detection of its content in plasma samples was put forward. We carried out an analysis of the plasma concentrations of procyanidin B2, procyanidin B3, catechin and epicatechin in normal and AD rats over time for determining the plasma concentration of GSP. Methods: We made use of 400 μL of methanol for the protein precipitation solvent in the plasma treatment. The chromatographic separation was carried out on a C18 column at a temperature of 20 °C. The mobile phase was a gradient of 0.1% formic acid in water and methanol within 15 min. Results: In the current research work, the plasma concentrations of procyanidin B2, procyanidin B3, catechin and epicatechin in AD rats were significantly higher as compared with those in normal rats (P < 0.05) and the content of epicatechin constituted the highest as compared with catechin, procyanidin B2 and procyanidin B3 following the administration of GSP. Conclusion: We discovered the better absorptions of these analytes in the AD group as compared with that in the normal group, providing an analytical basis for treating the AD with procyanidins.

Background: Generic products must be bioequivalent to the innovator brand product. Nevertheless, in addition to meeting bioequivalence standards, attention must be paid to the content of the active substance and contaminants in generic drugs. Objectives: This study compared the pharmaceutical quality of four generic losartan potassium formulations with the brand-name product: Cozaar®. Methods: The United States Pharmacopeia (USP) losartan potassium standard was used as reference material. The products tested (all 50 mg formulations) included four generic tablet formulations and the innovator brand product Cozaar®. Active substance content, organic impurities, and elemental impurities were assessed following the USP monograph for losartan potassium tablets and USP Chapter <233> on Elemental Impurities. Results: The results showed that three of the four generic products had low content of the active ingredient. The values ranged from 86.4 to 93.8%, being acceptable not below 95% of the labeled amount. Organic impurities were not detected in any of the products, and of the 13 elemental impurities tested, only four elements were detected. The elemental impurities Cr, Ni, Cu, and As were, however, in amounts within the limits established by the USP monograph. The only concern on the generic drugs analyzed was the low content of the active ingredient in 75% of the products. Conclusion: Since losartan is a drug of continuous use, lower content of the active ingredient may go unnoticed by the users of the generic product and entailed clinical consequences during long-term therapy.

Objective: To develop a reliable and sensitive high-performance liquid chromatographytandem mass spectrometry (HPLC-MS/MS) method for the quantification of selegiline in Beagle dog plasma and apply the validated method to study the pharmacokinetics and bioavailability of oral selegiline lyophilizate in Beagle dogs. Methods: Following alkalization with 1 M sodium hydroxide solution, selegiline and the Internal Standard (IS) zolmitriptan were extracted using tert-butyl methyl ether and separated on a CAPCELL PAK C18 column under isocratic conditions. They were detected by MS/MS using electrospray ionization (ESI) in the positive mode. Quantification was performed using multiple reaction monitoring (MRM) with transitions of m/z 188.05→90.9 for selegiline and m/z 288.05→57.95 for IS. Results: Calibration curves were constructed in the concentration range of 0.2–200 ng/mL with a lower limit of quantification (LLOQ) of 0.21 ng/mL. The matrix effect of dog plasma on the selegiline signal ranged from 98.8 to 105.6%, and the mean extraction recovery ranged from 79.0% to 81.4% at concentrations of 1.04, 20.8, and 166 ng/mL. The intra-day precision was lower than 6.86% and the inter-day precisions were lower than 4.63%. Conclusion: The validation results demonstrated the reliability of this bioanalytical method, which was successfully applied to study the pharmacokinetics and bioavailability of 1.25 mg of orally administered selegiline lyophilizate in Beagle dogs. The pharmacokinetic results were also compared with those obtained following intragastric (i.g.) and intravenous (i.v.) administration. Buccal delivery of selegiline was found to significantly increase its bioavailability.

Background: Desheng pills (DSP) consist of six traditional Chinese medicine. This preparation is used fornourishing blood, eliminating stasis, soothing liver and regulating menstruation, and can also be used to treat menoxenia and dysmenorrhea caus ed by qi stagnation and blood stasis. Objective: In this paper, an accurate and sensitive high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed and validated for simultaneous determination of seven active components (gallic acid, paeoniflorin, costunolide, dehydrocostuslactone, rutin, leonurine hydrochloride and ferulic acid) in the traditional Chinese formula-Desheng pills. Methods: The seven analytes were separated on Agilent ZORBAX SB-C18 column (250mm× 4.6mm, 5μm) maintained at the temperature of 30. Gradient elution was performed with the mobile phase of methanol (A)-0.1% phosphoric acid solution (B) at the flow rate of 1.0mL·min-1. The analysis was carried out at the wavelength of 225 nm, 256 nm, 277 nm and 320 nm with an injection volume of 10 μL. Results: The measured seven components showed good linear relationships within their own concentration ranges along with coefficients of determination ≥0.9996. The limits of detection and quantitation of all analytes were in the range of 0.19-13.51 μg/mL and 0.59-40.93 μg/mL, respectively. Average recoveries ranged from 98.82% to 102.01% with RSDs of 1.47%-1.99%. The content of tested components was in the range of 0.053-0.421 mg/g. Conclusion: The proposed method was found to be sensitive, accurate and reproducible, which provided an effective quantitative analytical method for quality control of Desheng pills.

Background: Individualized parenteral nutrition requires tailor-made preparation in hospital pharmacy followed by suitable quality control, including electrolytic content determination within complex and variable admixtures, containing up to 50 ingredients. Materials and Methods: An analytical tool has been developed to simultaneously quantify four major electrolytes in parenteral in parenteral nutrition mixtures (i.e., sodium, potassium, magnesium and calcium) using microwave plasma atomic emission spectroscopy (MP-AES). A comparison with reference inducted coupled plasma atomic emission spectroscopy (ICP-AES) was conducted. Calibration standard solutions ranged between 0.2 to 5.0 mg.L-1 for sodium, potassium and calcium and 0.1 to 2.5 mg.L-1 for magnesium. Wavelengths used were respectively 422.6 nm, 517.2 nm, 766.4 nm and 589.5 nm for calcium, magnesium, potassium and sodium, respectively. Accuracy profiles were used for the validation protocol. Results and Conclusion: The analytical protocol using MP-AES demonstrated high throughput and good sensitivity, thus enabling efficient quality control of manufactured individualized parenteral nutrition mixtures.

Introduction: A simple and reliable high performance liquid chromatographic method has been developed for the quantitative determination of pregabalin in bulk and dosage form. Pregabalin, a γ amino butyric acid analogue, has negligible sensitivity to UV or fluorescence detection. Hence, it has been derivatized by ninhydrin to form a chromophoric complex that could be quantified by UV detection. Materials and Methods: The concentration of ninhydrin was set to 5 mg/ml and a phosphate buffer solution (pH 7.4) was used as a solvent for the reaction. The resultant complex was separated by HPLC and detected by a UV detector at 569nm wavelength. Results: The developed method showed a linear response within 50 to 600 μg/mL of pregabalin. The method was accurate with mean recovery values within 100 ± 2%. The repeatability of the method was established by intra-day and inter-day precision study. Finally, a commercial pregabalin capsule was assayed by the developed HPLC method including ninhydrin derivatization. The result of the mean assay was found to be 100.37 ±2.94 %. Conclusion: This is the first time we are reporting pregabalin analysis using ninhydrin derivatization for HPLC analysis. Therefore, the developed method can be considered as a significant improvement in pregabalin quantitation and it can be easily applied for routine quality control tests of pregabalin.

Introduction: Capsaicin (8-methy-N-vanillyl-6-nonenamide), a potential analgesic derived from Capsicum annuum (Chili peppers), widely used from ancient times for its pharmacological activities such as anti-inflammatory, anti-oxidant and analgesic and provides relief from migraine and diabetes. But for obvious reasons, capsaicin cannot be administered directly. The present work was designed with a focus to comply with mandatory requirement in various pharmacopeias to know the actual content of API present in final formulations. The formulation (TS3) consisting of 3% lipid, with 4:6 ratio of the polymer and solvent, was found to be the optimized formulation, which gave the best evaluation with regard to the particle size (97.03±2.68) nm, polydispersity index (0.20±0.00), higher zeta potential (61.28±2.06) mv, morphological studies and highest drug entrapment efficiency (68.34±4.24)%. The prepared transferosome formulation was subjected to characterization by validated HP-TLC method consisting of N-Hexane: Tert- Iso-butyl-methyl ether in ratio (5:15) v/v. Linearity was performed in the range of 50-1500 ng/spot with LOD/LOQ 50 ng and 150 ng, with regression analysis (R) of 99.91%. Recovery analysis was performed at 3 different levels at 80, 100 and 120 with an average recovery of 106.97%, respectively. Till now, no analytical method has been reported, associated with the characterization of pharmaceutical nano-forms (Capsaicin), like transferosomes. Thus, the maiden validated HP-TLC method for concurrent analysis of capsaicin as API in nano-transferosome may be employed in process quality control of formulations containing the said API. Background: The irritability and adverse effects post application, leading to inflammation and neural pain at the site of administration of newly Capsaicin API and its chemical entities and marketed formulations are usually related to poor permeability, leading to drug complex reactions in the development phases or therapeutic failure along with the quantification of the same in blood plasma. However, advancement in drug formulations with the use of polymer: alcohol ratio and modernized analytical techniques for the quantification of Pharmaceutical APIs seems to be emerging and promising for overcoming pain and related inflammatory complications by formulating the APIs in Transferosome formulation with Validated HP-TLC technique being used as an effective economic and precise tool for quantitative analysis of APIs in their respective nano-forms. Objective: The study proposes a novel standardized method development and validation of pharmaceutical nanoforms with Capsaicin as API. Method: Capsaicin Transferosomes were formulated using Ultra probe sonication by utilizing different proportions of phospholipid 90G dissolved in a mixture of ethanol and propylene glycol. The formulation was subjected to Dynamic Light Scattering (DLS) technique for nano-particle analysis followed by characterization with respect to particle size, polydispersity index, zeta potential and entrapment efficiency. The morphological study of vesicles was determined using SEM and TEM. A Validated HP-TLC method for the identification and determination of Capsaicin in transferosomes formulation was performed as per the ICH guidelines. Results: The formulation gave the best evaluation for particle size (97.03±2.68) nm, polydispersity index (0.20±0.00), higher zeta potential (61.28±2.06) mv, morphological studies (SEM & TEM) and highest drug entrapment efficiency (68.34±4.24)%. DSC thermograms and FTIR spectral patterns confirmed no physical interaction by polymers with API. The prepared formulation was then characterized using HP-TLC method. The best resolution was found in NHexane: Tert-Isobutyl methyl ether in a ratio of 5:15 v/v. The Rf was found to be 0.3±0.03. Linearity was performed in a range of 50-1500 ng/spot, with regression analysis (R) of 99.91% Further, recovery analysis was done at 3 different levels as 80, 100 and 120 with an average recovery of 106.97%. The LOD/LOQ was found to be 50 and 150 ng, respectively. Precision was carried out in which % RSD was found to be precise and accurate. Conclusion: The outcomes of the present study suggested that the proposed novel formulation analyzed by Validated planar chromatographic technique (HP-TLC) for Capsaicin quantification in nanoforms may be employed as a routine quality control method for the said API in various other formulations.