Rapid on-site TLC-SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements.

A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC-SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS). The study showed that TLC-SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants.

The effects of contact lens materials on a multipurpose contact lens solution disinfection activity against Staphylococcus aureus.

OBJECTIVES: To determine the effect of 8 different lens materials on polyhexamethylene biguanide (PHMB) concentration in multipurpose solution (MPS) levels over time and to determine the effect of lenses on lens solution microbial efficacy over time. METHODS: Silicone hydrogel lenses and conventional hydrogel lenses were soaked in polypropylene lens cases filled with contact lens MPS containing 1 ppm PHMB for 6, 12, 24, 72, and 168 hours. Cases filled with the same solution without lenses were controls. After each time period, solutions from cases with the 8 types of lenses and controls were assayed for activity against Staphylococcus aureus according to International Organization for Standardization-14729 with modifications. Solutions were analyzed for PHMB concentration at each time point. RESULTS: Some of the different lens materials significantly affected the PHMB concentration (P<0.0001) and the biocidal efficacy. Etafilcon A lenses significantly decreased PHMB levels after only 6 hours of lens soak time. The product lot of MPS used was also significant (P<0.0001). Enfilcon A, senofilcon A, and lotrafilcon B lenses did not significantly decrease PHMB levels. CONCLUSIONS: The efficacy of MPS was affected by some lens materials and PHMB concentration. Lens materials differ in their effect on PHMB concentration and the subsequent efficacy of the MPS. Over time, some lens materials can significantly reduce the PHMB concentration and the MPS's microbial activity against S. aureus.

Impact of contact lens materials on multipurpose contact lens solution disinfection activity against Fusarium solani.

OBJECTIVE: To investigate the effects of eight different soft contact lenses on disinfection efficacy of a multipurpose solution (MPS) containing polyhexamethylene biguanide (PHMB) against Fusarium solani. METHODS: Six silicone hydrogel lenses (galyfilcon A, senofilcon A, comfilcon A, enfilcon A, balafilcon A, and lotrifilcon B) and two conventional hydrogel lenses (polymacon and etafilcon A) were placed in polypropylene lens cases filled with MPS containing 0.0001% PHMB and soaked for 6, 12, 24, 72, and 168 hours. After each interval, depleted MPS from lens cases were removed and assayed for activity against F. solani according to International Organization for Standardization (ISO) 14729 stand-alone procedure. A portion was aliquoted for chemical analysis. RESULTS: Soaking etafilcon A, balafilcon A, and polymacon lenses for 6 hours reduced the concentration of PHMB in MPS by more than half the stated labeled concentration, with concentrations below the limit of detection for etafilcon A-depleted and balafilcon A-depleted solutions after 12 and 72 hours of soaking, respectively. Except for comfilcon A-depleted solutions, all others failed to consistently obtain one log reduction of F. solani. The solutions soaked with etafilcon A, balafilcon A, and polymacon lenses for 24 hours or more lost all or almost all fungicidal activity against F. solani. CONCLUSIONS: Over time, the disinfectant uptake by some lenses can significantly reduce the PHMB concentration and the fungicidal activity of the MPS against F. solani. Current ISO methodology does not address the reduction in microbiocidal efficacy when lenses are soaked in MPS. The ISO committee should consider adding "soaking experiments" to quantify the effect that contact lens materials have on the performance of MPSs.

Environmental fate of polyhexamethylene biguanide.

Polyhexamethylene biguanide (PHMB) is used as a bacteriocidal agent in a variety of applications from medical devices to pools, but is highly toxic to some aquatic species. The stability of PHMB in various environmental matrices was examined. 80% of PHMB was present in fortified creek samples after 20 days, but bound immediately to soils with the exception of sandy soil. PHMB was absorbed to below detectable levels by weathered concrete within 12 h. In addition, one over the counter wound care product containing PHMB was evaluated to assess environmental leaching; detectable levels (20 µg/mL) were still present after 1 week.

Determination of Polyhexamethylene Biguanide Utilizing a Glucose Oxidase Enzymatic Reaction.

Polyhexamethylene biguanide (PHMB) is a cationic disinfectant widely used for personal-care products and for sanitizers in swimming pools. This paper describes a promotion effect of PHMB on a glucose oxidase (GOx) enzymatic reaction with ferricyanide ion and its analytical application. The promotion effect arose from a polyion complex formation between polycationic PHMB and polyanionic GOx. The promoted GOx reaction was analyzed by Michaelis-Menten equation, and the Michalis constant and catalytic constant were estimated to be 240 µM and 31 s-1, respectively. Utilizing the promotion effect, PHMB was successfully determined in the range of 0.05 to 0.40 ppm, and the detection limit was calculated to be 0.027 ppm. The visual detection and semi-determination of PHMB with the same concentration level were also possible. As an application, the method was applied to the determination of PHMB in a contact lens detergent and its model solution.

Analysis of polyhexamethylene biguanide and alexidine in contact lens solutions using capillary electrophoresis, ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry.

Polymeric biguanides, as well as quaternary ammonium compounds, are ubiquitous antimicrobial agents in healthcare. Due to the highly cationic and polymeric nature of these compounds and the complex matrices in which they are found, the analytical characterization of products containing them remains challenging. In this work an efficient, sensitive, and high-resolution separation protocol was developed to perform quantitative measurements (sub-mg L-1) of alexidine dihydrochloride (ADH) and polyhexamethylene biguanide (PHMB) in commercial multipurpose contact lens solutions (MPS). Initially, contactless conductivity (C4D) detection was explored, but lacked adequate selectivity and sensitivity to quantify PHMB or ADH in commercial MPS. To overcome these limitations, an alternative approach using solid phase extraction (SPE) followed by separation with reversed phase ultra-performance liquid chromatography (RP-UPLC) was developed for both ADH and PHMB separation and detection. The most sensitive and reliable method investigated utilized standard additions to compensate for matrix effects. For ADH, concentration values measured with the presented method were consistent with data provided by the MPS manufacturer (1.6 mg L-1) within 0.10 mg L-1. PHMB quantification in MPS products was successful at concentrations <1 mg L-1 with quantitative reproducibility better than 2% RSD. Comparison of blind sample testing using the RP-UPLC method showed strong correlation (R2 = 0.939) of PHMB concentrations with results obtained by the United States Food and Drug Administration using a published HPLC-Evaporative light scattering detection (ELSD) assay. A significant advantage of this method is the ability to partially resolve PHMB polydispersity, which to date has been minimally studied and explained. By coupling with electrospray mass spectrometry (MS), a general trend was observed for increased retention as a function of PHMB chain length. The improved robustness and reproducibility of UV detection versus ELSD coupled with the superior resolving power of UPLC is an asset to the detection and characterization of PHMB and ADH. In addition to quality control of MPS, this method has potential application to the analyses skin wipes, wound dressings and other medical products where understanding how manufacturing processes lead to differences in polydispersity is important to maximize the antimicrobial properties while minimizing toxicologic effects.

Potentiometric titration of polyhexamethylene biguanide hydrochloride with potassium poly(vinyl sulfate) solution using a cationic surfactant-selective electrode.

A potentiometric titration method using a cationic surfactant as an indicator cation and a plasticized poly(vinyl chloride) membrane electrode sensitive to the cationic surfactant is proposed for the determination of polyhexamethylene biguanide hydrochloride (PHMB-HCl), which is a cationic polyelectrolyte. A sample solution of PHMB-HCl containing an indicator cation (hexadecyltrimethylammonium ion, HTA) was titrated with a standard solution of an anionic polyelectrolyte, potassium poly(vinyl sulfate) (PVSK). The end-point was detected as a sharp potential change due to an abrupt decrease in the concentration of the indicator cation, HTA, which is caused by its association with PVSK. The effects of the concentrations of HTA ion and coexisting electrolytes in the sample solution on the degree of the potential change at the end-point were examined. A linear relationship between the concentration of PHMB-HCl and the end-point volume of the titrant exists in the concentration range from 2.0 x 10(-5) to 4.0 x 10(-4) M in the case that the concentration of HTA is 1.0 x 10(-5) M, and that from 1.0 x 10(-6) to 1.2 x 10(-5) M in the case that the concentration of HTA is 5.0 x 10(-6) M, respectively. The proposed method was applied to the determination of PHMB-HCl in some contact-lens detergents.

Salting-out assisted liquid-liquid extraction coupled with hydrophilic interaction chromatography for the determination of biguanides in biological and environmental samples.

A new salting-out assisted liquid-liquid extraction (SALLE) sample preparation method for the determination of the polar anti-diabetic biguanide drugs (metformin, buformin and phenformin) in blood plasma, urine and lake water samples were developed. The SALLE was performed by mixing samples (plasma (0.2mL), urine or lake water (1.0mL)) with acetonitrile (0.4mL for plasma, 0.5mL for urine or lake water), sodium hydroxide powder was then added for the phase separation. The effects of type of salting-out reagent, type of extraction solvent, volumes of acetonitrile and sample, amount of sodium hydroxide, vortexing and centrifugation times on the extraction efficiency were investigated. The upper layer, containing the biguanides, was directly injected into a HPLC unit using ZIC-HILIC column (150mm×2.1mm×3.5µm) and was detected at 236nm. The method was validated and calibration curves were linear with r2>0.99 over the range of 20-2000µgL-1 for plasma and 5-2000µgL-1 for urine and lake water samples. The limits of detection were in the range (3.8-5.6)µgL-1, (0.8-1.5)µgL-1 and (0.3-0.8)µgL-1 for plasma, urine and lake water, respectively. The accuracies in the three matrices were within 87.3-103%, 87.4-109%, 82.2-109% of the nominal concentration for metformin, buformin and phenformin, respectively. The relative standard deviation for inter- and intra -day precision were in the range of 1.0-17% for all analytes in the three matrices.

Simultaneous measurement of proguanil and its metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography, and its preliminary application in relation to genetically determined S-mephenytoin 4'-hydroxylation status.

A simple high-performance liquid chromatographic (HPLC) assay method was developed for the measurement of proguanil (PG) and its major metabolites, cycloguanil (CG) and 4-chlorophenyl-biguanide (CPB), in human plasma and urine. The assay allowed the simultaneous determination of all analytes in 1 ml of plasma or 0.1 ml of urine. The detection limits of PG, CG, and CPB, defined as the signal-to-noise ratio of 3, were 1 and 5 ng/ml for plasma and urine samples, respectively. Recoveries of the analytes and the internal standard (pyrimethamine) were > 62% from plasma and > 77% from urine. Intra-assay and interassay coefficients of variation for all analytes in plasma and urine were < 10% except for the values of CG and CPB, which ranged from 10% to 15% at one or two concentrations among 4-5 concentrations studied. The clinical applicability of the method was assessed by the preliminary pharmacokinetic study of PG, CG, and CPB in six healthy volunteers with the individually known phenotypes (extensive and poor metabolizers) of S-mephenytoin 4'-hydroxylation, suggesting that individuals with a poor metabolizer phenotype of S-mephenytoin have a much lower capacity to bioactivate PG to CG compared with the extensive metabolizers.

Analysis of proguanil and its metabolites by application of the sweeping technique in micellar electrokinetic chromatography.

The method of applying large sample volumes in micellar electrokinetic chromatography termed sweeping is applied to determine the conservative limits of detection of some basic drugs in plasma and urine. The biguanides proguanil, 4-chlorophenylbiguanide and cycloguanil are used as models of basic drugs and the limits of detection obtained compared with those previously reported for capillary zone electrophoresis using field-amplified sample injection (FASI) and also by LC using off-line preconcentration. It is found that the sweeping method can be applied to extracts of such biological matrices. The limits of detection obtained by sweeping are improved over FASI for plasma but not for urine and the limits of detection are higher than those reported for LC, for these compounds.

Conversion of biguanides into substituted s-triazines assayable by GC or mass fragmentography.

Substituted s-triazines were prepared by the treatment of biguanides with various organic acid anhydrides. This reaction permits the ready conversion of the hypoglycemic drugs phenformin, buformin, and metformin and of other analogous biguanides into compounds suitable for GC and mass fragmentographic determination with a high degree of sensitivity. Mass spectral data and Kováts retention indexes are presented for all s-triazines prepared for this study.

Gravimetric determination of chlorhexidine using tetraphenylborate ion.

A precise and accurate gravimetric procedure was developed for the determination of chlorhexidine diacetate, digluconate, or dihydrochloride. Sodium tetraphenylborate solution was the precipitant in an acidic medium (pH 1). Tablets containing both chlorhexidine diacetate and benzocaine also were assayed.

Gas chromatographic determination of buformin in body fluids and tissues, using a nitrogen phosphorus detector: application to a postmortem case.

A specific method is described for the quantitation of the oral hypoglycemic biguanide buformin in biological material by gas chromatography. Phenformin, another biguanide derivative, is used as internal standard. Both compounds are converted to the corresponding s-triazine derivatives by reacting with acetic anhydride prior to gas chromatography with nitrogen-specific detection. The described procedure has been applied to the quantitative assay of buformin in body fluids and tissues, obtained from a postmortem case. The results of these toxicological investigations are discussed.

Determination of biguanide groups in polyhexamethylene biguanide hydrochloride by titrimetric methods.

The biguanide concentration of polyhexamethylene biguanide hydrochloride (PHMB-HCl) was measured by non-aqueous titration with HClO4, argentometric titration, the Kjeldhal method, and colloidal titration. The summation value of non-aqueous titration and argentometric titration corresponded to two titrable nitrogens in five nitrogens per one unit of PHMB-HCl, and consisted with the result of the Kjeldhal method to the five nitrogens. The colloidal titration of PHMB-HCl at pH 2.05 was equal to that with the two nitrogens. The relative standard deviations of non-aqueous titration, argentometric titration, the Kjeldhal method, and colloidal titration were 0.50% for 8 runs, 0.13% for 7 runs, 3.61% for 6 runs, and 0.69% for 6 runs, respectively.

Synthesis of morpholinobiguanide derivatives.

Several new morpholinobiguanide derivatives were synthesized to be tested as hypoglycemic agents, by condensing morpholinobiguanide with alkyl or aryl isocyanate and isothiocyanate, and with dialkylaminoalkyl chloride. The carbamate, prepared from morpholinobiguanide and ethyl chloroformate, was condensed with different aryl amines and certain sulpha drugs. Most of the prepared compounds are easily soluble in water and preliminary examinations as hypoglycemics show promising results.

Biguanide related compounds in traditional antidiabetic functional foods.

Biguanides such as metformin are widely used worldwide for the treatment of type-2 diabetes. The identification of guanidine and related compounds in French lilac plant (Galega officinalis L.) led to the development of biguanides. Despite of their plant origin, biguanides have not been reported in plants. The objective of this study was to quantify biguanide related compounds (BRCs) in experimentally or clinically substantiated antidiabetic functional plant foods and potatoes. The corrected results of the Voges-Proskauer (V-P) assay suggest that the highest amounts of BRCs are present in green curry leaves (Murraya koenigii (L.) Sprengel) followed by fenugreek seeds (Trigonella foenum-graecum L.), green bitter gourd (Momordica charantia Descourt.), and potato (Solanum tuberosum L.). Whereas, garlic (Allium sativum L.), and sweet potato (Ipomea batatas (L.) Lam.) contain negligible amounts of BRCs. In addition, the possible biosynthetic routes of biguanide in these plant foods are discussed.

Determination of polyhexamethylene biguanide hydrochloride using photometric colloidal titration with crystal violet as a color indicator.

A solution of polyhexamethylene biguanide hydrochloride (PHMB-HCl) was titrated with a standard solution of potassium poly(vinyl sulfate) (PVSK) using crystal violet (CV) as an photometric indicator cation. The end point was detected by a sharp absorbance change due to an abrupt decrease in the concentration of CV. A linear relationship between the concentration of PHMB-HCl and the end-point volume of the titrant existed in the concentration range from 2 to 10 × 10(-6) eq mol L(-1). Back-titration was based on adding an excess amount of PVSK to a sample solution containing CV, which was titrated with a standard solution of poly(diallyldimethylammonium chloride) (PDADMAC). The calibration curve of the PHMB-HCl concentration to the end point volume of the titrant was also linear in the concentration range from 2 to 8 × 10(-6) eq mol L(-1). Both photometric titrations were applied to the determination of PHMB-HCl in a few contact-lens detergents. Back-titration showed a clear end point, but direct titration showed an unclear end point. The results of the back-titration of PHMB-HCl were compared with the content registered in its labels.