Oral Intake of Enzymatically Decomposed AP Collagen Peptides Improves Skin Moisture and Ceramide and Natural Moisturizing Factor Contents in the Stratum Corneum.

The stratum corneum (SC) is the outermost layer of the epidermis and plays an important role in maintaining skin moisture and protecting the skin from the external environment. Ceramide and natural moisturizing factor (NMF) are the major SC components that maintain skin moisture. In this study, we investigated whether the oral intake of enzymatically decomposed AP collagen peptides (APCPs) can improve skin moisture and barrier function by assessing changes in the ceramide and NMF contents in the SC after APCP ingestion with the aim to develop a skin functional food. Fifty participants orally ingested APCP (1000 mg) or placebo for 12 weeks, and then, skin hydration and skin texture were evaluated. SC samples were collected to analyze skin scaling, ceramide, and NMF contents. Participants in the APCP group exhibited improved skin moisture content by 7.33% (p = 0.031) and roughness by -4.09% (p = 0.036) when compared with those in the placebo group. NMF content; the amounts of amino acids (AA), including glycine and proline; and AA derivatives were significantly increased in the APCP group (31.98 µg/mg protein) compared to those in the placebo group (-16.01 µg/mg protein) (p = 0.006). The amounts of total ceramides and ceramide subclasses were significantly higher in the APCP group than in the placebo group (p = 0.014). In conclusion, our results demonstrate that APCP intake improves skin moisture and increase the ceramide and NMF contents in the SC, thereby enhancing the skin barrier function.

Development and validation of UPLC method for WST-1 cell viability assay and its application to MCTT HCE™ eye irritation test for colorful substances.

WST-1 [Water Soluble Tetrazolium-1; 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt)] is widely used in the cell viability assays replacing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A water-soluble formazan dye (4-[1-(4-Iodophenyl)-5-(4-nitrophenyl)formaz-3-yl]-1,3-benzene disulfonate, disodium salt) is produced from the reduction of WST-1 tetrazolium, of which optical density at 450 nm is measured to evaluate cell viability. Colorful substances may interfere with spectrometric measurement, and a method to specifically detect WST-1 formazan is required. Here, a simple, rapid, sensitive, and specific ultra-performance liquid chromatography coupled to UV detector (UPLC-UV) was developed and validated for the WST-1 formazan. For the application to cell viability assay, the supernatant from WST-1 assay was injected without sample preparation procedure and a single run was completed within 5 min. Chromatographic separation was achieved on BEH C18 column (1.7 µm, 2.1 × 50 mm) using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 2.5-120 µg/mL WST-1 formazan, which encompasses WST-1 formazan concentrations from 2% cell viability to 2 fold of 100% cell viability. The intra- and inter-day precisions were measured to be below 5% and accuracies were within the range of 91.8-104.9%. The validated method was successfully applied to the test of colorful substances in vitro eye irritation test with a human cornea-like epithelium, and in vitro cytotoxicity in HaCaT, human keratinocyte cell line.

Skin irritation and sensitization potential of oxidative hair dye substances evaluated with in vitro, in chemico and in silico test methods.

Permanent oxidative hair dyes are widely used but their toxicity is not well-established. Here we aimed to evaluate the skin sensitization and irritation of nine hair dye substances (MAP, MRP-N, RS, PAOX, 2,4-DAPE, 2,6-PYR, PPD, Grey HED and PM) permitted for use in EU and Korea, using in vitro and in chemico and in silico test methods. Skin sensitization was evaluated by the KeratinoSens™ assay, Direct Peptide Reactivity Assay (DPRA) and DEREK. Six of nine dyes tested were determined as sensitizers in common. However, the decision for MAP, RS or PAOX was diverged across assays showing 2 positives and 1 negative. Skin irritation of hair dye substances was assessed with or without 6% H2O2 on a reconstructed human epidermis, Epiderm™, which demonstrated that H2O2 increased the skin irritation potential of some hair dyes. PPD and PM were determined to be irritants with H2O2. Epidermal damages by hair dye and H2O2 could be further confirmed through the histology of tissue remaining after MTT assay. Collectively, our study demonstrated that hair dyes possess potential skin sensitization and irritation issues which could be further aggravated by H2O2.

Quercetin and fisetin enhanced the small intestine cellular uptake and plasma levels of epi-catechins in in vitro and in vivo models.

Quercetin and fisetin, known as catechol-containing flavonoids, could positively affect the absorption of catechins due to their strong affinity for catechol-O-methyl transferase (COMT), which can methylate and cause the excretion of catechins. The current study examined the effect of quercetin and fisetin on the absorption of epi-catechins (ECs) by using a Caco-2 cell line and an in vivo model. The intestinal transport of total catechins by Caco-2 cells was enhanced from 1.3- to 1.6-fold and 1.4- to 1.7-fold by adding quercetin and fisetin, respectively, compared to the control. It was even higher in the treatment with a mixture of quercetin and fisetin. While EC had the highest value of intestinal transport (169% of the control) in 10% quercetin treatment, EGC (235%), EGCG (244%), and ECG (242%) were significantly transported in the treatment with a 5% mixture of quercetin and fisetin (p < 0.05). In an in vivo pharmacokinetic study, the values of the area under the plasma concentration-time curve (AUC, ng h mL-1) were also higher in rats orally administered EGCG with 10% quercetin (365.5 ± 25.5) or 10% fisetin (825.3 ± 46.7) than in those administered EGCG only (111.3 ± 13.1). Methylated quercetin and methylated fisetin were determined to be m/z 317.24 and m/z 301.25 [M + H]+ with their own product ions, respectively. The results indicate that quercetin or fisetin is superior to ECs for methylation by COMT.

Nervonoylceramide (C24:1Cer), a lipid biomarker for ocular irritants released from the 3D reconstructed human cornea-like epithelium, MCTT HCE™.

Due to invasive and painful procedures during in vivo rabbit eye irritation test, in vitro alternative methods have been widely investigated. Recently, 3D reconstructed human cornea-like epitheliums (RhCEs) garner a huge attention. RhCEs employ the tissue viability as a primary endpoint to determine ocular irritancy but additional biomarkers may improve its predictive capacity. Here, we explored lipid biomarkers for ocular irritants in MCTT HCE™ RhCE model. Three irritants; sodium lauryl sulfate, benzalkonium chloride and triton X-100 were selected to represent anionic, cationic and non-ionic detergent respectively. After treating MCTT HCE™ with irritants, the alteration of lipids in the treated tissues was examined with Nile Red staining, which revealed the depletion of corneal lipids. We further quantitated the release of ceramides and free fatty acids, major lipid components of cornea, into the medium during the post-treatment incubation, employing a sensitive UPLC-MS/MS method. Among 44 lipid species, nervonoylceramide (C24:1Cer) was found to be released commonly by all three irritants in a concentration-dependent manner. Tests with 10 additional reference substances further supported that C24:1Cer release was significantly correlated with viability. Examination of the genes involved in the biosynthetic pathway for C24:1Cer revealed that stearoylCoA desaturase (SCD) and elongase1 (ELOVL1) were upregulated, suggesting that lipids and related genes may be employed as biomarkers for ocular irritants.

Influence of flavonol-rich excipient food (onion peel and Dendropanax morbifera) on the bioavailability of green tea epicatechins in vitro and in vivo.

The impacts of onion peel (OP) and Dendropanax morbifera (DM), as excipient foods rich in flavonols, on the digestive recovery, intestinal absorption, and pharmacokinetics of GT epicatechins were studied via an in vitro digestion model system with Caco-2 cells and an in vivo study. The digestive stability of total epicatechins recovered from GT upon the addition of 2% DM was up to 1.12 times higher than that observed with OP. The combined effects of OP and DM, which were observed with 2% OP + DM in a ratio of 1 : 4 (w : w), significantly increased (by a factor of 1.31) the digestive recovery of total epicatechins (p < 0.05). Remarkable cellular uptakes of EC (185.36%) and ECG (188.08%) were found with 4% OP + DM (4 : 1, w : w), and those of EGC (112.30%) and EGCG (136.27%) were obtained with 2% OP + DM (4 : 1, w : w) and 1% OP + DM (1 : 1, w : w), respectively. The peak plasma concentrations of total epicatechins from GT, GT + 5% OP, GT + 5% DM, and GT + 2% OP + 2% DM were 1044.78 ± 609.10, 2267.18 ± 3734.38, 1270.35 ± 547.59, and 714.53 ± 499.27 ng mL-1, respectively. The Cmax value of total epicatechins in rats orally administrated with GT with 5% OP was found to be approximately twice of that obtained with GT alone. The co-ingestion of GT with flavonol-rich excipient foods possibly enhances the absorption of epicatechins because flavonols act as not only enhancers of digestive stability but also modulators of the biotransformation of epicatechins. The results obtained from the current study suggest that the absorption of GT catechins can vary depending upon the kinds and doses of excipient foods co-ingested.

Increased cis-to-trans urocanic acid ratio in the skin of chronic spontaneous urticaria patients.

Increased filaggrin expression was found to be correlated with severity scores in chronic spontaneous urticaria (CSU); however, the role of filaggrin breakdown products (FBPs) in CSU has not been studied. We collected stratum corneum (SC) specimens from the volar forearms of 10 CSU patients, 10 AD patients, and 10 healthy normal controls (NCs) and measured contents of FBPs (pyrrolidone carboxylic acid [PCA] and urocanic acid [UCA]) using UPLC-MS/MS, transepidermal water loss (TEWL) and epidermal pH. Compared to NCs, cis-UCA level was increased in CSU lesions (P < 0.05) and decreased in AD lesions (P < 0.01). The cis-to-trans-UCA ratio in SC specimens from CSU patients was significantly greater than those from AD and NC subjects. AD lesions had lower FBP and PCA contents compared to NC skin (both P < 0.001), and higher TEWL and pH compared to CSU lesions. Moreover, cis-UCA, but not trans-UCA, enhanced the IgE-mediated basophil activation, as well as IgE- and calcium-mediated degranulation of LAD-2 cells, in a dose-dependent manner. These findings suggest that increased cis-to-trans UCA ratio in the epidermis is a distinct feature of CSU, which could enhance mast cell degranulation. Modulation of cis-UCA may be a potential target for skin diseases associated with IgE-mediated mast cell degranulation.

Determination of N-nitrosodiethanolamine, NDELA in cosmetic ingredients and products by mixed mode solid phase extraction and UPLC-tandem mass spectrometry with porous graphitic carbon column through systemic sample pre-cleanup procedure.

A rapid, sensitive, accurate and specific ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the detection of N-nitrosodiethanolamine (NDELA), a highly toxic contaminant in cosmetic raw materials and products was developed and validated. Systematized sample preparation steps were developed according to product types. Various SPE cartridges and columns were examined to establish the condition of SPE and chromatographic separation for NDELA. Sample cleanup steps consisting of solvent and liquid-liquid extraction tailored to the various sample matrix types were established prior to mixed mode SPE (Bond Elut AccuCAT). Chromatographic separation was achieved within 7 min on a porous graphitic carbon (PGC) column using a gradient elution with the mobile phase of 1mM ammonium acetate containing 0.1% acetic acid and methanol. NDELA was monitored using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode (m/z 134.9>103.7(quantifier) and 73.7(qualifier ion)) with d8-NDELA (m/z 143.1>111.0) as internal standard. The standard curves were linear over the concentration range of 1-100 ng/mL with a correlation coefficient higher than 0.99. The limit of detection (LOD) and the limit of quantification (LOQ) was 10 and 20 µg/kg, respectively (0.5 and 1 ng/mL in standard solution). The intra- and inter-day precisions were estimated to be below 11.1% and accuracies were within the range of 90.8-115.8%. The validated method was successfully applied to the analysis of real samples including raw materials, skin care, make-up, shampoos and hair products.

A rapid and highly sensitive UPLC-MS/MS method using pre-column derivatization with 2-picolylamine for intravenous and percutaneous pharmacokinetics of valproic acid in rats.

A rapid, highly sensitive and specific ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for the detection of valproic acid (VPA) in rat plasma following the topical application was developed and validated. This method was carried out with pre-column derivatization using 2-picolylamine (PA) which reacts with the carboxylic acid group of VPA. The derivatization was completed in 10min and the resulting PA-VPA derivative enabled the sensitive detection of VPA in selected reaction monitoring (SRM) mode. Sample preparation was done with simple liquid-liquid extraction and chromatographic separation was achieved within 5min on a C18 column using a gradient elution with the mobile phase of 2mM ammonium formate containing 0.1% formic acid and methanol. The standard curves were linear over the concentration range of 0.07-200µg/mL with a correlation coefficient higher than 0.99. The limit of detection (LOD) and the lower limit of quantification (LLOQ) was 0.03 and 0.07µg/mL, respectively with 100µL of plasma sample. The intra- and inter-day precisions were measured to be below 10.7% and accuracies were within the range of 94.1-115.9%. The validated method was successfully applied to the pharmacokinetics of VPA in the rat following topical and intravenous applications.

Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin-myosin Va interaction.

Human skin hyperpigmentation disorders occur when the synthesis and/or distribution of melanin increases. The distribution of melanin in the skin is achieved by melanosome transport and transfer. The transport of melanosomes, the organelles where melanin is made, in a melanocyte precedes the transfer of the melanosomes to a keratinocyte. Therefore, hyperpigmentation can be regulated by decreasing melanosome transport. In this study, we found that an extract of Saururus chinensis Baill (ESCB) and one of its components, manassantin B, inhibited melanosome transport in Melan-a melanocytes and normal human melanocytes (NHMs). Manassantin B disturbed melanosome transport by disrupting the interaction between melanophilin and myosin Va. Manassantin B is neither a direct nor an indirect inhibitor of tyrosinase. The total melanin content was not reduced when melanosome transport was inhibited in a Melan-a melanocyte monoculture by manassantin B. Manassantin B decreased melanin content only when Melan-a melanocytes were co-cultured with SP-1 keratinocytes or stimulated by α-MSH. Therefore, we propose that specific inhibitors of melanosome transport, such as manassantin B, are potential candidate or lead compounds for the development of agents to treat undesirable hyperpigmentation of the skin.

Rapid, simultaneous and nanomolar determination of pyroglutamic acid and cis-/trans-urocanic acid in human stratum corneum by hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization tandem mass spectrometry.

A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0-250 ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5 ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2 ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3-102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans-urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.

Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method.

PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C(18) column (4.6 mm × 75 mm, 3.5 µm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492→156; IS, 506→156, m/z). The calibration curve was linear over the range of 1.0-500 ng/ml (r(2)>0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ± 15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze-thaw cycles and 4-week storage in a freezer at -80°C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1h and 3.8h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application (F% 0.2-1.7%).

Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid.

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

Determination of spatial distribution of melamine-cyanuric acid crystals in rat kidney tissue by histology and imaging matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry.

After the outbreak of acute renal failure associated with melamine-contaminated pet food, many attempts have been made to uncover the mechanism underlying the renal toxicity caused by melamine and melamine-related compounds. Using rat models, we investigated the renal crystal formation following the ingestion of a melamine-cyanuric acid mixture (M+CA, 1:1) to gain insight into the M+CA-induced renal toxicity. M+CA did not induce toxicity in precision-cut kidney slices, suggesting that M+CA does not have a direct nephrotoxicity. On the contrary, oral administration of M+CA for 3 days induced nephrotoxicity as determined by increased serum blood urea nitrogen and creatinine, reduced creatinine clearance, and enlarged kidneys in the animals treated with 50 mg/kg M+CA (melamine, 25 mg/kg, and cyanuric acid, 25 mg/kg; 2 of 10 animals) and 100 mg/kg M+CA (9 of 9 animals). While urine crystals were found in all animals treated with M+CA (25-100 mg/kg), histological examination revealed that renal crystals could be observed only in the kidneys of animals showing signs of nephrotoxicity. Remarkably, at 50 mg/kg M+CA, crystals were observed mainly in the medulla region of the kidney, while at 100 mg/kg, crystals were disseminated throughout the cortex and medulla regions. To further investigate the crystal formation by M+CA, matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) imaging mass spectrometry detecting melamine distribution through monitoring the product ion (m/z 85, M + H) from melamine (m/z 127, M + H) was developed to directly obtain the image of melamine distribution in the kidney. The distribution image of melamine in kidney tissue confirmed that dense points of melamine were located only in the medulla region at 50 mg/kg M+CA, while at 100 mg/kg, they were disseminated widely from the cortex to medulla. These results demonstrated that M+CA ingestion could lead to crystal formation in kidney tubules along the osmotic gradient and that renal crystal formation is closely linked with M+CA-induced nephrotoxicity.

Pharmacokinetic study of ginsenoside Re with pure ginsenoside Re and ginseng berry extracts in mouse using ultra performance liquid chromatography/mass spectrometric method.

Ginsenoside Re is the major ginsenoside in ginseng berry(GB) extract and its pharmacokinetics were studied following the intravenous and oral administration of pure Re or ginseng berry extract in mouse with doses of 10 and 50 mg/kg using ultra performance liquid chromatography mass spectrometric (UPLC/MS) method which can simultaneously determine ginsenoside Re, Rg1 and Rh1 in mouse serum. The serum samples were pretreated by protein precipitation and chromatographic separation was performed on AQUITY UPLC BEH C(18) column using gradient elution with the mobile phase of 5 mM ammonium formate and acetonitrile. Analytes and digoxin (I.S.) were analyzed and identified using an electrospray negative ionization mass spectrometry in the selected ion monitoring mode with the linear concentration range of 5.0-5000 ng/mL and lower limits of detection (LLOD) under 2.5 ng/mL. Ginsenoside Re was rapidly cleared from the body with a short half-life (0.2+/-0.03 h for male and 0.5+/-0.08 h for female mice after i.v.) and oral absorption was generally poor (F% 0.19-0.28). Notably, GB extract showed a superior oral absorption of ginsenoside Re (F% 0.33-0.75) at equivalent ginsenoside Re dose to pure ginsenoside Re, indicating that GB extract might be a good form for ginsenoside Re intake.

Simultaneous determination of two Amadori compounds in Korean red ginseng (Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection.

A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng (Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15-102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.

Determination of hexavalent chromium in cosmetic products by ion chromatography and postcolumn derivatization.

Chromium hydroxide green [Cr(2)O(OH)(4)] and chromium oxide green (Cr(2)O(3)) are colouring agents for use in cosmetic products. These colourants may contain chromium (VI), which cause skin allergies through percutaneous adsorption on the skin. Eye shadow is a representative cosmetic product in which significant colourants are used. We analysed the chromium (VI) in the eye shadows by ion chromatography and post column derivatization. We optimize conditions of chromium (VI) analysis in eye shadows. During the pretreatment procedure, there are no exchange of chromium (III) to chromium (VI). This method has a limit of quantification for chromium (VI) of 1.0 microg l(-1), recovery rate of 100 +/- 3% and analysis time less than 10 min. This result is 300 times more sensitive than the high-performance liquid chromatography method. We applied the optimized method to analyse 22 eye shadows and 6 colouring agents. 2 out of 22 of the products contained more than 5 mg l(-1). In our previous work, 5 mg l(-1) of Cr represented a threshold level. There was much more Cr(VI) in the colouring agents. The Cr(VI) in one of the colouring agents was 97.6 mg l(-1).