LC-MS bioanalytical method for simultaneous determination of latanoprost and minoxidil in the skin.

The association of minoxidil sulphate and latanoprost is currently emerging as a promising strategy for the treatment of androgenic alopecia, which is the most common type of scalp hair loss. In order to support the development of new pharmaceutical products containing such drugs combination, this study proposes a simple and efficient LC-MS bioanalytical method to simultaneously quantify minoxidil sulphate and latanoprost in different skin layers. Compounds separation was performed by liquid chromatography using a C18 column with gradient elution of a mobile phase composed of 0.1 % formic acid in acetonitrile and water at a flow rate of 0.5 mL min-1. Determinations were executed using mass spectrometry equipped with an ESI interface operating in a positive ionization mode. Quantification was performed using selective ion mode monitoring of m/z 210.1 for minoxidil sulphate and 433.3 for latanoprost. The matrix effect was very pronounced in samples containing some skin layers or electrolyte solution. Accordingly, a calibration curve for each contaminant group was built, leading to correlation coefficient values higher than 0.99. Additionally, lower limits of detection and quantification were obtained, and precision (repeatability and intermediate precision) achieved results with a coefficient of variation less than 15 %. Drug recovery from skin samples was higher than 70 %, fulfilling the recommendations. Also, the bioanalytical method was successfully tested in in vitro skin penetration studies proving its effectiveness in the development of topical formulations containing both drugs.

Development and validation of rapid and simultaneous method for determination of 12 hair-growth compounds in adulterated products by UHPLC-MS/MS.

Synthetic hair-growth compounds have been illegally used in diverse products to enhance the short-term efficacy of these products. In this study, a rapid and simultaneous method for the determination of hair-growth compounds in adulterated products based on ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. The limit of detection (LOD) and limit of quantitation (LOQs) of the method were 0.08-43.6ng/mL and 0.27-145ng/mL for the solid-, liquid-, and cream-type samples, respectively. Good calibration linearity for all compounds was demonstrated with a correlation coefficient (r2) higher than 0.997. The intra- and inter-assay precisions were within 11%. The corresponding accuracies were 86-117% and 81-113%, respectively. The mean recoveries obtained for the solid-, liquid, and cream-type samples ranged from 87 to 114%, with a relative standard deviation (RSD) within 6%. The RSD of the stability evaluated at 4°C for 48h was less than 6%. The established method was used to screen 76 samples advertised as hair-growth treatments, from online and offline markets, over the course of two years. In 10% of the samples, four compounds, including triaminodil, minoxidil, finasteride, methyltestosterone, and testosterone-propionate were detected. The concentrations were in the range of 0.5-16.4mg/g. This technique provides a reliable platform for technical analysis for continuous monitoring of adulterated products to protect public health.

Stability of an extemporaneously compounded minoxidil oral suspension.

PURPOSE: Results of a study to determine the stability of an extemporaneously compounded minoxidil oral suspension under various temperature and stress conditions are reported. METHODS: Commercially available minoxidil tablets (10 mg) were crushed to a fine powder, and predetermined amounts of 2 suspending vehicles were added to produce a 1-mg/mL suspension, which was stored in glass bottles at room temperature (25 ± 2 °C) or in a refrigerator (4 ± 2 °C). To simulate daily patient use, 5 days weekly 1 bottle of the suspension was removed from refrigerated storage and shaken and 0.5 mL of the contents discarded. At each specified time point, samples were analyzed in duplicate (n = 6 for each test condition) using a validated high-performance liquid chromatography method. Samples were visually observed and their pH measured at each time point. Microbiological studies were performed on day 0 and at week 24. RESULTS: The mean percentage of initial minoxidil concentration remaining in all refrigerated samples exceeded 90% throughout the 24-week study, with no change in appearance, pH, microbial activity, odor, or redispersibility. During storage at room temperature, the suspension exhibited a color change at week 4, with slight sedimentation after 6 weeks, although minoxidil recovery exceeded 90% for 10 weeks. CONCLUSION: An extemporaneously compounded minoxidil oral suspension was stable for 24 weeks when stored in a refrigerator. This suspension can be used for up to 3 weeks when stored at room temperature.

Determination of illegal adulteration of dietary supplements with synthetic hair-growth compounds by UPLC and LC-Q-TOF/MS.

In this study, we developed a UPLC-PDA and LC-Q-TOF/MS method to identify and measure the following prohibited substances that may be found in dietary supplements:triaminodil, minoxidil, bimatoprost, alimemazine, diphenylcyclopropenone, α-tradiol, finasteride, methyltestosterone, spironolatone, flutamide, cyproterone, dutasteride, and testosterone 17-propionate.The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, accuracy, precision, LOD, LOQ, recovery, and stability. The method was completely validated showing satisfactory data for all method validation parameters. The linearity was good (R2 > 0.999) with intra- and inter-day precision values of 0.2-3.4% and 0.3-2.9%, respectively. Moreover, the intra- and inter-day accuracies were 87-102% and 86-103%, respectively, and the precision was better than 9.4% (relative standard deviation).Hence, the proposed method is precise and has high quality,and can be utilised to comprehensively and continually monitor illegal drug adulteration in various forms of dietary supplements. Furthermore, to evaluate the applicability of the proposed method, we analysed 13 hair-growth compounds in 78 samples including food and dietary supplements. Minoxidil and triaminodil were detected in capsules at concentrations of 4.69 mg/g and 6.54 mg/g. In addition, finasteride was detected in a tablet at 13.45 mg/g. In addition, the major characteristic fragment ions were confirmed once again using LC-Q-TOF/MS for higher accuracy.

Stability indicating RP-HPLC method development and validation for the simultaneous determination of aminexil and minoxidil in pharmaceutical dosage form.

The objective of the present work is to develop stability indicating high-performance liquid chromatographic method for the simultaneous determination of aminexil and minoxidil in pharmaceutical dosage form. The chromatographic separation was achieved with BDS Hypersil C18 column (250 mm×4.6 mm×5 µ) as stationary phase and phosphate buffer and acetonitrile (78:22) as mobile phase. The method was employed by using a flow rate of 1.1 mL/min kept at 30°C. The detection wavelength was kept at 238 nm by using photo-diode array detector. The retention times of the aminexil and minoxidil were found to be 2.3 min and 3.9 min, respectively. The method developed was validated in accordance with ICH guidelines with respect to the stability indicating capacity of the method including system suitability, accuracy, precision, linearity, range, limit of detection, limit of quantification and robustness. The linearity responses of aminexil and minoxidil were found to be in the concentration ranges of 18.75-112.5 µg/mL and 25-150 µg/mL, respectively. The LOD and LOQ values for aminexil were found to be 0.31 and 0.92 µg/mL and minoxidil were found to be 0.03 and 0.10 µg/mL respectively. The percentage recoveries for both the drugs were found in the range of 98-101%. This method is accurate, precise and sensitive; hence, it can be employed for routine quality control of aminexil and minoxidil in pharmaceutical industries and drug testing laboratories.

Electrochemical determination of minoxidil in different pharmaceutical formulations by flow injection analysis.

The electrochemical behavior and amperometric-FIA quantification of minoxidil at a glassy carbon electrode is described. The procedure is based on electrochemical oxidation at 0.800 V (vs. Ag/AgCl/NaCl(3 M) in a phosphate buffer solution. Minoxidil was determined over the range 1 x 10(-7) - 1 x 10(-4) M. Different analytical parameters and electrode stability were analyzed to obtain the best electrode performance. The optimal conditions were: working potentials, 0.800 V; flow rate, 0.74 mL min(-1); and solution pH 7.0. This system allowed a sampling rate of 120 samples per hour without any pretreatment. The proposed method was used for minoxidil quantification in pharmaceutical preparations with satisfactory results. The accuracy of FIA-amperometric method was established by a comparison with the conventional UV determination technique using a paired t-test indicating the absence of systematic errors.

A method for the determination of minoxidil in hair-regrowth formulations by micellar electrokinetic capillary chromatography.

A method based on micellar electrokinetic capillary chromatography (MEKC) was developed for determination of minoxidil in Rogaine and competing products. The original intent of the work was to offer an orthogonal means to HPLC for testing illicit imitations of Rogaine. However, because the patent has since expired, we offer the procedure as a confirmatory measure to HPLC for assay of generic minoxidil products. The MEKC procedure complements an earlier method based on free solution capillary electrophoresis (FSCE), designed to the same end. Validation was carried out on both a Dionex CES-1, which utilizes gravity injection, and a PE-ABI 270HT, which employs vacuum injection. The procedure was validated for both active pharmaceutical ingredient and for minoxidil solutions. The run buffer is pH 7.0, 20 mM sodium phosphate, 20 mM sodium dodecyl sulfate, with 10% isopropanol; the internal standard is dl-tryptophan. The method bears the attributes of simplicity, ease of use, and short analysis time (12 min). It is selective with respect to known process and degradation impurities. High efficiency was achieved on the CES-1, with a plate count exceeding 200,000 for minoxidil at an elution time of 9 min. Although slight differences in performance were noted across the two instruments, results on both were in conformance with modern day validation expectations. Comparison of MEKC with HPLC resulted in slightly higher values for the former, but all results met registration specifications and internal targets.

Development and validation of a procedure for the determination of minoxidil in hair-regrowth formulations using two variants of capillary zone electrophoresis.

A high performance capillary electrophoresis method was developed and validated for purity assessment of minoxidil bulk drug and for determination of minoxidil in Rogaine. The principal use of the method was in analyzing illicit minoxidil-containing hair-regrowth samples. Although validated for Rogaine, the procedure proved equally viable on illicit minoxidil-containing preparations. The developed method fulfilled the goal of providing an orthogonal technique to HPLC for confirmation of the presence of minoxidil in these imitations. The method was validated on two instruments, one utilizing EK injection, the other gravity injection. It is selective for minoxidil, which is separated from known process impurities and the single degradation impurity. Validation figures of merit for linearity/recovery (accuracy) and precision were in accordance with current expectations for method validation.

Rapid determination of trace amounts of minoxidil in hamster skin follicles with various formulations using narrow-bore LC/EC.

A sensitive liquid chromatographic method with electrochemistry (LC/EC) was developed for the determination of trace of minoxidil in hamster skin follicles after topical administration of the ear using various formulations. The minoxidil in the sebaceous glands of the hamster ear was isolated from the skin and the follicles in different skin layers were treated with aqueous trichloroacetic acid followed by acetonitrile. The supernatant was directly injected into the LC/EC system and minoxidil was detected by oxidation at +800 mV versus Ag/AgCl using a glassy carbon electrode. The analytical recoveries were between 94.4 and 103.1% and the linearity was excellent up to 250 microg/ml with a regression coefficient (r(2)) of 0.9988. The LC/EC and the widely used radiolabeled scintillation methods agree well and both show high sensitivities. The LC/EC method is rapid and cost-effective with a detection limit of only 1 ng/ml.

Sulfation of minoxidil by human liver phenol sulfotransferase.

The N,O-sulfate of minoxidil (Mnx) is the active agent in producing the vasodilation and the hair-growth stimulating responses observed with Mnx treatment. In this report, Mnx sulfation activity was assayed in cytosol prepared from several normal human livers, and Mnx sulfation was shown to correlate significantly with the activity of the phenol-sulfating form of phenol sulfotransferase (P-PST) activity in the same livers. No correlation was observed between Mnx sulfation and the dopamine or dehydroepiandrosterone (DHEA) sulfotransferase activities present in human liver. Mnx sulfation also copurified with P-PST activity during the purification of P-PST from human liver. During the purification procedure, Mnx and p-nitrophenol sulfotransferase (P-PST) activities were resolved from the dopamine and DHEA sulfation activities catalyzed by the monoamine-sulfating form of phenol sulfotransferase (M-PST) and DHEA sulfotransferase respectively. Also, purified DHEA sulfotransferase was not capable of sulfating Mnx, and no data were obtained to indicate that Mnx is a substrate for M-PST. p-Nitrophenol, a substrate for P-PST, was demonstrated to be a competitive inhibitor of Mnx sulfation catalyzed by purified P-PST when Mnx was the variable substrate. These results indicate that Mnx is sulfated and, therefore, bioactivated by P-PST in human liver.

Determination of minoxidil in pharmaceutical formulations by difference spectrophotometry and liquid chromatography (HPLC).

A simple difference spectrophotometric method has been developed for the selective and rapid determination of minoxidil in pharmaceutical formulations. The method is based on the characteristic modifications of the minoxidil UV spectrum induced by pH changes; a difference spectrum can be obtained and the absorbance A282.8 or the absorbance difference delta A = (A282.8-A259.2) can be used for the selective drug quantitation. As an alternative, a derivative second-order spectrophotometric method was also developed. The spectrophotometric methods were applied to the quality control of commercial minoxidil dosage forms and the results were comparable with those obtained by a reference HPLC procedure.

Determination of minoxidil in bulk drug and pharmaceutical formulations by ion-pairing high-performance liquid chromatography.

An ion-pairing liquid chromatographic method with UV detection is described for the determination of minoxidil in bulk drug, compressed tablet, and topical solution formulations. The chromatographic system consists of a microparticulate octadecylsilica column and a mobile phase composed of sodium dioctylsulfosuccinate in aqueous methanol (pH 3). The bulk drug and the topical solution samples are prepared by the dissolution of the drug in internal standard solution. Sample preparation for the compressed tablet formulation involves dissolving the drug from an aliquot of pulverized sample and centrifuging to remove insoluble excipients. Quantitative recovery of minoxidil from formulation excipients was demonstrated; assay precision was less than 1% CV.

Radioimmunoassay of minoxidil in human serum.

A simple, sensitive, and specific radioimmunoassay for determining minoxidil was developed. Antiserums to two minoxidil haptens were compared for cross-reactivity and assay levels on human serums. One antiserum had little cross-reactivity with minoxidil metabolites. The radioimmunoassay is specific for determining minoxidil directly in serum without extraction. Human serum minoxidil levels were determined from a single oral dose.