UPLC-TQD-MS/MS Method Validation for Quality Control of Alkaloid Content in Lepidium meyenii (Maca)-Containing Food and Dietary Supplements.

Lepidium meyenii Walp. (Brassicaceae), also known as Maca or Peruvian ginseng, is a common ingredient in food supplements with many claimed health benefits, such as improved endurance, increased energy level, and enhanced sexual properties. Due to potential toxicity of its chemicals, including alkaloids, some regulatory authorities, e.g., in Belgium, Germany, the United States, expressed concerns about the safe consumption of Maca root. However, due to the lack of commercial standards, no established analytical method currently exists for this purpose. The current project focuses on the quantitative determination of potentially toxic alkaloids from Maca. The current study presents the first analytical method for quality control of alkaloid content in Maca-containing food and dietary supplements, assessing the presence of 11 major compounds belonging to three different classes, i.e., imidazole, ß-carboline, and pyrrole alkaloids. An accurate, rapid, and sensitive UPLC-TQD-MS/MS method is reported, which was fully validated according to the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and SANTE/11312/2021 guidelines. To ensure the method's applicability and practicability in the absence of primary standards, validation of secondary standards (SSs) alongside primary standards (PSs) was also conducted for imidazole alkaloids. As a result, in Maca raw powder, total alkaloid content was found to vary from 418 to 554 ppm (mg/kg). Furthermore, all quantified imidazole alkaloids were ascertained to be the major alkaloids with the total content from 323 to 470 ppm in Maca raw powder, followed by the ß-carboline and pyrrole alkaloids. It was also observed that the commercial preparation of finished products affects the total alkaloid content, evidenced by the large variation from 56 to 598 ppm. Ultimately, from a regulatory point of view, it seems advisible not to request the complete absence of the alkaloids but to impose a maximum level based on safety considerations. In addition to the analytical method, a low-cost, simple, and scalable synthetic scheme of macapyrrolins A, C, and G was reported for the first time.

Isocyanates may contribute to allergic contact dermatitis from diabetes devices and wound dressings.

BACKGROUND: Isocyanates are well-known occupational allergens, but can also be present in medical devices. OBJECTIVES: To highlight that contact sensitization to isocyanates might contribute to allergic contact dermatitis (ACD) from polyurethane (PU)-containing diabetes devices and wound dressings. PATIENTS AND METHODS: Nineteen patients with suspected ACD from diabetes devices and/or wound dressings were patch tested to an isocyanate series. Four wound dressings, six diabetes devices and four monomeric isocyanate patch test preparations were analysed with gas chromatography - mass spectrometry. RESULTS: Eight patients reacted to isocyanates and corresponding amines: 3 to isophorone diisocyanate (IPDI), 4 to 4,4'-diaminodiphenylmethane (MDA), 4 to 2,4-toluene diisocyanate (TDI) and 1 to polymeric methylene diphenyl diisocyanate (PMDI). Three of four wound dressings contained isocyanates (methylene diphenyl diisocyanate [MDI], TDI and/or IPDI), whereas five of six diabetes devices contained 4,4'-MDI, and one of them also IPDI. None of the medical devices contained 1,6-hexamethylene diisocyanate. Contrary to IPDI, and especially MDI, only the concentration of the TDI patch test preparation corresponded approximately (80%) to its label. CONCLUSION: Patch tests with isocyanates may be worth-while in patients with suspected ACD from PU-containing medical devices. Besides MDA, and PMDI, also TDI might potentially be a marker for MDI-sensitization.

Allergic contact dermatitis from ("hypoallergenic") adhesives containing D-limonene.

BACKGROUND: Besides being a potential component of (some species of) colophonium, D-limonene is also used as a tackifier in the production of adhesives. Hydroperoxides of limonene are well-known skin sensitizers. OBJECTIVES: To show that D-limonene may be present in colophonium-containing but also colophonium-free ("hypoallergenic") adhesives, and that patients suffering from allergic contact dermatitis (ACD) from both types of adhesives might display positive patch test reactions to limonene hydroperoxides in this regard. METHODS: Five patients with suspected ACD from adhesives were patch tested to the baseline series (containing limonene hydroperoxides 0.3 and 0.2% pet.), additional series and, if available, to the culprit adhesives. The adhesives labelled as containing colophonium (n = 3) or free from it (n = 2) were analysed with gas chromatography - mass spectrometry (GC-MS) for the presence of D-limonene. RESULTS: All five patients sensitised to adhesives had (strong) positive patch test reactions to limonene hydroperoxides. The presence of D-limonene, and/or related components, could be demonstrated in all three colophonium-containing and, surprisingly, also in two colophonium-free ("hypoallergenic") tapes. CONCLUSIONS: D-limonene may be present in both regular and "hypoallergenic" adhesives, with limonene hydroperoxides potentially contributing to ACD from such medical devices. The use of fragrance chemicals in adhesives deserves further research.

Identification and Quantification of Polymethoxylated Flavonoids in Different Citrus Species Using UPLC-QTOF-MS/MS and HPLC-DAD.

Many species from the genus Citrus are used in traditional medicine and contain polymethoxylated flavonoids. These compounds show anti-inflammatory and chemopreventive activities, among others, and therefore have a big potential to be developed as therapeutic agents or dietary supplements. Citrus species are different in their profile and yield of polymethoxylated flavonoids. Therefore, polymethoxylated flavonoids were identified and quantified in seven different Citrus species, including wild-type and commercially available species. All species were profiled using UPLC-QTOF-MS/MS analysis combined with mass spectral molecular networking. A total of 38 polymethoxylated flavonoids were detected and 8 of them were present in every species. As the yield of polymethoxylated flavonoids was different for each species, a generally applicable HPLC-diode array detection method was developed and validated according to the ICH guidelines to quantify the amount of nobiletin and the total amount of polymethoxylated flavonoids expressed as nobiletin. Analysis of the seven samples showed evidence that wild-type Citrus species (e.g., Citrus depressa) contain higher yields of polymethoxylated flavonoids compared to commercially available species (e.g., Citrus limon). Qualitative analysis revealed the broadest variety of different PMFs in C. depressa, Citrus reticulata, and Citrus reticulata × Citrus sinensis, which makes them interesting sources of polymethoxylated flavonoids for future development as therapeutic agents or dietary supplements.

The presence of benzophenone in sunscreens and cosmetics containing the organic UV filter octocrylene: A laboratory study.

BACKGROUND: The reason why patients photosensitized to the drug ketoprofen (KP) may develop severe photoallergic skin reactions to octocrylene (OCT), an organic ultraviolet filter in sunscreens and cosmetics, remains largely unknown. OCT can be synthesized by using unsubstituted benzophenone (BP), a possible human carcinogen. OBJECTIVES: To verify if, and to what extent, BP residues are present in OCT-containing consumer products. METHODS: The raw material of OCT and 39 skincare products, of which 28 contain OCT, were chemically analysed for the presence of BP by means of liquid chromatography. RESULTS: In the OCT raw material and in all 28 OCT-containing products the presence of BP could be demonstrated, mostly in concentrations above 10 ppm (0.001%), whereas a majority of OCT-free products (8/11, 73%) did not contain BP. Moreover, BP concentrations significantly increased, in a time- and temperature-dependent manner, likely due to the additional degradation of OCT. CONCLUSIONS: Photoallergic contact dermatitis from OCT in patients photosensitized to KP might rely on residual BP impurities. Toxicological and ecological studies that evaluate the safety of OCT might also need to consider the concomitant presence of BP.

In vitro antigenotoxic activity, in silico ADME prediction and protective effects against aflatoxin B1 induced hepatotoxicity in rats of an Erythrina latissima stem bark extract.

Stem bark of Erythrina latissima E. Mey (Leguminosae) contains a wide range of prenylated flavonoids able to counteract the genotoxic properties of aflatoxin B1 (AFB1). Thus, the hypothesis was raised that E. latissima stem bark extracts (ELBE) may counteract the in vivo hepatotoxic effects of aflatoxins, contaminants in food and feed. An HPLC-DAD method was developed and validated to determine the level of flavonoid aglycones (11.82%) and glycosides (16.17%). ADME, pharmacokinetic and drug-likeness assessment of major flavonoids of ELBE, using the web tool SwissADME, showed good oral bioavailability. The protective effect of ELBE against AFB1 induced genotoxicity in the Vitotox assay after metabolic activation was confirmed (IC50 of 44.32 µg/ml), followed by evaluation of its inhibitory effect on hepatotoxicity in rats induced by the same agent. Male Wistar rats were orally treated with ELBE (20 mg/kg, 50 mg/kg and 100 mg/kg) or curcumin (500 mg/kg) combined with piperine (20 mg/kg) - positive control, for 8 days prior to AFB1 exposure (1 mg/kg). The ELBE group showed a decreased activity of ALP and γ-GT compared to the AFB1 group. Histopathological examination of the liver demonstrated ameliorative effects of ELBE. Thus, ELBE could have a protective effect against hepatotoxins such as AFB1.

Ultrasound-assisted extraction optimization and validation of an HPLC-DAD method for the quantification of polyphenols in leaf extracts of Cecropia species.

Cecropia species are traditionally used in Latin American folk medicine and are available as food supplements with little information warranting their quality. The optimum conditions for the extraction of chlorogenic acid (CA), total flavonoids (TF) and flavonolignans (FL) from leaves of Cecropia species were determined using a fractional factorial design (FFD) and a central composite design (CCD). A reversed-phase high-performance liquid chromatographic method coupled to a diode array detector (HPLC-DAD) was validated for the quantification of CA, TF and FL, following the ICH guidelines. Quantitative and Principal Component Analysis (PCA) was also performed. The extraction-optimization methodology enabled us developing an appropriate extraction process with a time-efficient execution of experiments. The experimental values agreed with those predicted, thus indicating suitability of the proposed model. The validation parameters for all chemical markers of the quantification method were satisfactory. The results revealed that the method had excellent selectivity, linearity, precision (repeatability and intermediate precision were below than 2 and 5%, respectively) and accuracy (98-102%). The limits of detection and quantification were at nanogram per milliliter (ng/mL) level. In conclusion, the simultaneous quantification of chemical markers using the proposed method is an appropriate approach for species discrimination and quality evaluation of Cecropia sp.

Toll-Like Receptor-Dependent Immunomodulatory Activity of Pycnogenol®.

BACKGROUND: Pycnogenol® (PYC), an extract of French maritime pine bark, is widely used as a dietary supplement. PYC has been shown to exert anti-inflammatory actions via inhibiting the Toll-like receptor 4 (TLR4) pathway. However, the role of the other receptors from the TLR family in the immunomodulatory activity of PYC has not been described so far. AIM: The aim of this study was to investigate whether PYC might exert its immunomodulatory properties through cell membrane TLRs (TLR1/2, TLR5, and TLR2/6) other than TLR4. Moreover, the effect of gastrointestinal metabolism on the immunomodulatory effects of PYC was investigated. FINDINGS: We showed that intact non-metabolized PYC dose-dependently acts as an agonist of TLR1/2 and TLR2/6 and as a partial agonist of TLR5. PYC on its own does not agonize or antagonize TLR4. However, after the formation of complexes with lipopolysaccharides (LPS), it is a potent activator of TLR4 signaling. Gastrointestinal metabolism of PYC revealed the immunosuppressive potential of the retentate fraction against TLR1/2 and TLR2/6 when compared to the control fraction containing microbiota and enzymes only. The dialyzed fraction containing PYC metabolites revealed the capacity to induce anti-inflammatory IL-10 secretion. Finally, microbially metabolized PYC affected the colonic microbiota composition during in vitro gastrointestinal digestion. CONCLUSIONS: This study showed that gastrointestinal metabolism of PYC reveals its biological activity as a potential inhibitor of TLRs signaling. The results suggest that metabolized PYC acts as a partial agonist of TLR1/2 and TLR2/6 in the presence of the microbiota-derived TLR agonists (retentate fraction) and that it possesses anti-inflammatory potential reflected by the induction of IL-10 from THP-1 macrophages (dialysate fraction).

Advantages of a validated UPLC-MS/MS standard addition method for the quantification of A-type dimeric and trimeric proanthocyanidins in cranberry extracts in comparison with well-known quantification methods.

The berries of Vaccinium macrocarpon, cranberry, are widely used for the prevention of urinary tract infections. This species contains A-type proanthocyanidins (PACs), which intervene in the initial phase of the development of urinary tract infections by preventing the adherence of Escherichia coli by their P-type fimbriae to uroepithelial cells. Unfortunately, the existing clinical studies used different cranberry preparations, which were poorly standardized. Because of this, the results were hard to compare, which led sometimes to conflicting results. Currently, PACs are quantified using the rather non-specific spectrophotometric 4-dimethylaminocinnamaldehyde (DMAC) method. In addition, a normal phase HPTLC-densitometric method, a HPLC-UV method and three LC-MS/MS methods for quantification of procyanidin A2 were recently published. All these methods contain some shortcomings and errors. Hence, the development and validation of a fast and sensitive standard addition LC-MS/MS method for the simultaneous quantification of A-type dimers and trimers in a cranberry dry extract was carried out. A linear calibration model could be adopted for dimers and, after logaritmic transformation, for trimers. The maximal interday and interconcentration precision was found to be 4.86% and 4.28% for procyanidin A2, and 5.61% and 7.65% for trimeric PACs, which are all acceptable values for an analytical method using LC-MS/MS. In addition, twelve different cranberry extracts were analyzed by means of the newly validated method and other widely used methods. There appeared to be an enormous variation in dimeric and trimeric PAC content. Comparison of these results with LC-MS/MS analysis without standard addition showed the presence of matrix effects for some of the extracts and proved the necessity of standard addition. A comparison of the well-known and widely used DMAC method, the butanol-HCl assay and this newly developed LC-MS/MS method clearly indicated the need for a reliable method able to quantify A-type PACs, which are considered to be the pharmacologically active constituents of cranberry, since neither the DMAC or butanol-HCl assays are capable of distinguishing between A and B-type PACs and therefore cannot detect adulterations with, for example, extracts with a high B-type PAC content. Hence, the combination of the DMAC method or butanol-HCl assay with this more specific LC-MS/MS assay could overcome these shortcomings.

Development and validation of a robust high-performance liquid chromatographic method for the analysis of monacolins in red yeast rice.

A robust analytical method, using reversed phase high-performance liquid chromatography with diode array detection, was developed and validated for the quantification of monacolins in red yeast rice bulk products. Tests on the composition of the extraction solvent, extraction time and the number of repetitions of extraction were evaluated with the aim of complete extraction of the monacolins and minimal transitions between the monacolins during analysis. Monacolin K (acid form), monacolin K (lactone form) and minor monacolin peaks were separated on a C18 column (250×4.6mm, 5µm) using acetonitrile/0.1% trifluoroacetic acid as the mobile phase. For the calibration curve of monacolin K (lactone form), a linear correlation in the range 6-119µg/mL was found. The precision of the method for time and concentration gave a relative standard deviation of less than 5%, which was deemed acceptable. The recovery of the method was 98.75%.

Quantification of xanthohumol, isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin in hop extracts and derived capsules using secondary standards.

Hop is a well-known and already frequently used estrogenic phytotherapeutic, containing the interesting prenylflavonoids, xanthohumol (XN), isoxanthohumol (IXN), 8- and 6-prenylnaringenin (8-PN and 6-PN). Since the use of secondary standards can form a solution whenever the determination is required of certain components, not commercially available or too expensive, it was decided to develop an accessible HPLC-DAD method for the determination of these prenylflavonoids. The amounts were determined in hop extract and capsules, using quercetin and naringenin as secondary standards. After optimization of the sample preparation and HPLC conditions, the analysis was validated according to the ICH guidelines. The response function of XN, 8-PN, quercetin and naringenin showed a linear relationship. For the determination of XN, a calibration line of at least three concentrations of quercetin has to be constructed. The correction factors for XN (quercetin) and for 8-PN (naringenin) were validated and determined to be 0.583 for XN, and 1.296 for IXN, 8-PN and 6-PN. The intermediate precision was investigated and it could be concluded that the standard deviation of the method was equal considering time and concentration (RSD of 2.5-5%). By means of a recovery experiment, it was proven that the method is accurate (recoveries of 96.1-100.1%). Additionally, by analysing preparations containing hop extracts on the Belgian market, it was shown that the method is suitable for its use, namely the determination of XN, IXN, 8-PN and 6-PN in hop extract and capsules, using quercetin and naringenin as secondary standards.

Densitometric thin-layer chromatographic determination of aescin in a herbal medicinal product containing Aesculus and Vitis dry extracts.

A thin-layer chromatographic (TLC) method is developed to analyze the total saponin content, also referred to as the aescin content, in a herbal medicinal product (HMP) containing two dry extracts in capsules. The capsules contain 250 mg of Aesculus hippocastanum dry extract, 120 mg of Vitis vinifera dry extract and 50mg of excipients. After a purification step using C(18) solid phase extraction (SPE) cartridges, the samples are analyzed on a silica-gel HPTLC plate with the upper layer of a mixture of acetic acid/water/butanol (10/40/50 v/v/v) as the mobile phase. Spots are visualized by spraying with anisaldehyde reagent and heating the plate for 5-10 min (100-105 degrees C) and measured at a wavelength of 535 nm. This method, applicable for the quality control and stability investigation of both the Aesculus dry extract and HMP capsules thereof containing Vitis dry extract in combination with the Aesculus dry extract, is validated according to the International Conference on Harmonization (ICH) guidelines. The proposed assay method is specific for aescin in the presence of Vitis dry extract and formulation excipients. Analysis of stressed samples in forced degradation tests proves the method to be applicable for stability evaluation. The standard aescin curve is linear (r > 0.99) over a concentration range of 0.16-0.80 microg/spot. Recovery from the HMP capsules is statistically equal to 100%. The precision of the method with respect to time and concentration is acceptable, with relative standard deviation (RSD) values of 1.28 and 1.49%, respectively.

Quality control of roots of Eleutherococcus senticosus by HPLC.

An HPLC method based on several known methods for the determination of eleutherosides B and E was developed, optimised and validated in terms of linearity, precision (repeatability and intermediate precision on different days and at different concentration levels) and accuracy (recovery). The extraction procedure, the extraction solvent and the extraction yield were evaluated and optimised. A reversed-phase RP-18 column gradient eluted with a two-phase system consisting of phosphoric acid:water (0.5:99.5) and acetonitrile was used to evaluate the samples; detection was at 220 nm. Although eleutherosides B and E are commercially available, they are very costly, and therefore ferulic acid was chosen as external standard. The correction factors for the response of ferulic acid against both eleutherosides were determined and validated. This method, accepted by the European Pharmacopoeia Commission, will be included in the monograph on Eleutherococcus senticosus roots to assay the content of eleutherosides B and E.

Fast high-performance liquid chromatography method for quality control of soy extracts.

Soy extracts contain a mixture of isoflavones belonging to the group of phytoestrogens. In the quality control of soy the amount of isoflavones, both aglycones and glycosides, is usually determined by means of reversed-phase HPLC-UV. On conventional C18-material columns, long analysis times are required in order to separate this complex mixture. In order to speed up analysis, the separation was optimized using two linked monolithic silica-based reversed-phase C18 columns. A spectacular decrease of the analysis time, i.e. almost three-fold, was achieved by applying a flow rate of 3-4 ml/min without loosing any separation efficiency. This analysis method for determination of isoflavones in soy extracts in less than 25 min was fully validated according to the ICH guidelines.