Issue 5 • 2015 Riedel-de Haën®

Kromega® Certified Reference Materials by Jaytee® pg. 3

HPLC Instrument Calibration

Phytochemical Standards

Marine Biotoxin CRMs

Carotenoid Standards

Glucuronide Drug Testing

BAM-Certified Reference Materials

High-Quality HPLC Buffers

Water Monitoring in Cosmetics Feature Article Sigma-Aldrich®’s Collaboration – Quality and Trust 3 Kromega® Certified Reference Materials by Jaytee® Now Available at Sigma-Aldrich® Dr. Magdalena Ulman Standards 5 Phytochemical Standards New Products and New Brochure Available Dear Reader, 6 Marine Biotoxin CRMs Products from the National Research Council of Canada (NRC) Sigma-Aldrich® works continually to develop long-term Now Available through Sigma-Aldrich® relationships with suppliers who demonstrate excellent and consistent product quality. This includes not only reagents 8 Isomeric Distribution of Astaxanthin and standards, but also consumables needed for different analytical techniques. New Standards for all-trans, 9-cis and 13-cis Astaxanthin Our partners, such as Hellma® or Specac®, have already proven their outstanding 12 Traceability Means Reliability performance level and consulting experience in the areas of UV/VIS, fluorescence New Organic TraceCERT® CRMs for Chromatography and IR spectroscopy. This issue presents one of our newest important partners, who 14 Glucuronide Drug Testing Applications deliver innovative products and dependable service. by Mass Spectrometry Jaytee® is well respected among the UK’s largest pharmaceutical and chemical companies Certified Spiking Solutions® of Glucuronides as a quality lead supplier and service provider. The kromega range of Qualification Labware Standards for HPLC and UV Spectrophotometers is designed to provide you with a solid foundation for your HPLC and UV spectrophotometer instrumentation qualification. 16 New Hellma® TrayCell™ – Photometric Micro Volume Analysis in the Nanoliter Range The feature article in this issue focuses on Jaytee’s kromega standards and gives insight DNA/RNA – Simply Analyzed into this high-quality product group. 17 Timestrip® – The Smart Way to Measure Time and Temperature Kind regards, Chromatography 19 Reversed-phase HPLC Buffers High-quality Buffers (Solutions, Solids or Concentrates) Titration Dr. Magdalena Ulman Product Manager Analytical Reagents 21 Water Determination in Cosmetic Products [email protected] Karl Fischer Titration with Hydranal® Reagents

Analytix is published five times per year by Sigma-Aldrich Chemie GmbH, Industriestrasse 25, CH-9471 Buchs SG, Switzerland Publisher: Sigma-Aldrich Marketing Operations Europe Publication Manager: Michael Jeitziner Editor: Daniel Vogler sigma-aldrich.com/analytix 2 Back to Table of Contents

Feature Article 3

Kromega® Certified Reference Materials by Jaytee® Now Available at Sigma-Aldrich®

Paul Boother, Operations Manager Regulatory [email protected] Any laboratory using analytical instrumentation is required to provide Magdalena Ulman, Product Manager Analytical Reagents [email protected] a form of instrument calibration as proof the hardware is operating correctly. The USP has a monograph [1] which defines how the equipment qualification process can be managed. The EDQM has published guidelines [2] on the suitable qualification tests for both HPLC and GC, while the EP has a monograph [3] devoted to the qualification of UV-visible spectrophotometers. In addition, the FDA requires that any instrument qualification should be based upon the needs of the user, not on the claims of the manufacturer. This suggests that it is wise to periodically check the performance of the instrument. Jaytee’s experience in supporting highly regulated customers has resulted in countless audits of our qualification services by inspectors from the FDA and MHRA, as well as by the quality departments of numerous blue chip pharmaceutical companies. This has allowed us to fine-tune the kromega CRMs to be a repeatable, reliable product which is quickly becoming the industry standard for qualification. In addition, analysts can be assured that their Introduction instrumentation is operating correctly when it successfully passes user-defined acceptance criteria using the kromega CRMs. This Reliable measurement is the basis for all analytical techniques. is because the kromega range is independently produced and Analysts spend a vast amount of time validating methods, running therefore gives a valuable “second opinion” rather than relying on system suitability tests and performing QC checks. All this is based on using the manufacturer’s non-accredited standard, analyzed with the the rationale that the instrument must be working correctly because manufacturer’s software and passing the manufacturer’s criteria. all of the above was successful, but what if the instrument itself was faulty? The truth is that many methods are developed using instrumentation which has not been proven to work correctly since the time it was installed in the laboratory. Jaytee has almost 30 years of experience supporting analytical science and has grown along with the highly regulated industries, such as pharmaceuticals and biotechnology, to deliver high quality instrument qualification services. Jaytee is now the only company in the world accredited by UKAS to IEC – ISO 17025 for the calibration of HPLC, GC and UV/visible spectrophotometers. In order to achieve this, Jaytee developed the kromega Certified Reference Material (CRM) range which provides the CRMs required to measure critical instrument parameters, thereby demonstrating that the instruments are operating to the level required by the laboratory. This range is accredited to ISO Guide 34 by UKAS, and is therefore the “Gold Standard” of Certified Reference Materials, being of the same standing as the renowned Sigma-Aldrich TraceCERT® range of CRMs. Not only do the kromega CRMs have a higher level of accreditation than any of the instrument manufacturers, but they are also suitable for cross-platform use, allowing a baseline of testing between the various OEMs. This means analysts can be assured of the level of accuracy of both their new UHPLC and the 15 year old HPLC residing in the same laboratory.

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4 Analytix | 5: 2015

Data Quality Triangle The Kromega Product Range The USP monograph [1] discusses the need for data quality to be The current product range covers a variety of HPLC (or UHPLC) CRMs underpinned at every level, as demonstrated in the Data Quality suitable for calibrating systems with any style of UV detection (e.g., Triangle. The concept is simple in that lower levels of the triangle must MWD, DAD, PDA, etc.) and a range of UV/Visible Spectroscopy CRMs, be complete, otherwise anything above is questionable. To explain, which meet the European Pharmacopeia’s requirements for the if the HPLC detector wavelength is inaccurate or the autosampler calibration of UV/Vis spectrophotometers. is injecting half the expected volume, then any method validation Future additions to the range will include GC FID and other will be flawed as the conclusions will be based on inaccurate results. HPLC detectors. It follows that the system suitability will also be inaccurate and any quality control samples worthless. Unfortunately, it is also likely that Kromega CRMs for HPLC: any subsequent method transfer will then be problematic even when trying to move to an instrument of identical configuration. Part Number Description Z803898 HPLC Autosampler If the instrument were then serviced and the issue inadvertently fixed, Z804002 UV Absorbance Linearity the method may no longer perform as it did previously because the results would then be accurate. It may exhibit increased or decreased Z804126 UV Low Wavelength absorbance values which may well be outside the validated range of Z804223 UV Multi Wavelength the method. Z804339 HPLC Qualification Kit Kromega CRMs for UV/Visible Spectrophotometers: Part Number Description Z804452 UV Photometric Accuracy Z804568 UV Resolution (EP) Z804665 UV Stray Light (EP) Z804789 UV Spec Qualification Kit If you would like to obtain further information about kromega CRMs available at Sigma-Aldrich, please visit sigma-aldrich.com/jaytee

References [1] USP <1058> Analytical Instrument Qualification. [2] EDQM PA/PH/OMCL (11) 04, Qualification of Equipment, Annex 1: Qualification of HPLC Equipment. [3] European Pharmacopeia 2.2.25 Absorption Spectrophotometry, Ultraviolet and Visible.

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FeatureStandards Article 5

Phytochemical Standards New Products and New Brochure Available

Matthias Nold, Product Manager Analytical Standards Cat. No. Description Package Size [email protected] 44787 Aconitine 10 mg 55659 Aspidinol 10 mg 72795 Butein 10 mg 61221 Castalagin 10 mg 40820 Chebulinic acid 10 mg 39273 Coptisin chloride 10 mg 43046 Cyanidin 3-sambubioside chloride 5 mg 42739 Cyanidin 3-sophoroside chloride 5mg 56523 Cycloolivil 10 mg 42575 Dehydrocostus lactone 10 mg 42883 Delphinidin 3-sambubioside chloride 5 mg 52559 Eupatorin 10 mg 94334 Formononetin 50 mg 42924 Germacrone 10 mg 89960 Ginkgotoxin 10 mg 94381 Ginsenoside R0 10 mg 44987 Ginsenoside Rc 5 mg 43019 Ginsenoside Rg6 5 mg Pharmaceutical products of plant origin, so called herbal medicinal 43084 Ginsenoside Rh3 5 mg products, enjoy great popularity as an alternative or complement to 42754 Ginsenoside Rk1 5 mg synthetic drugs. 43534 Glycitein 10 mg As the composition of a plant may vary depending on the time and 94821 Guaiaverin 5 mg location of the harvest, the concentrations of active ingredients and 42891 Hyperforin (dicyclohexylammonium) salt 5 mg other characteristic components should be monitored to ensure 55193 Marmelosin 10 mg product safety and quality consistency. 75032 Methyl trans-cinnamate 50 mg Sigma-Aldrich® offers analytical standards as well as primary reference 42904 Oxypeucedanin 5 mg standards of active ingredients and marker compounds of medicinal 40525 Picroside I 10 mg plants. This portfolio comprises over 500 products and is continually 68922 Picroside II 10 mg expanded. 79899 Piperitone 10 mg On our webpage at sigma-aldrich.com/medicinalplants an up-to- 07267 Protopine 10 mg date list of these products can be found. 00290 (−)-Sinigrin hydrate 10 mg In addition, we recently issued a new comprehensive brochure 53382 Skullcapflavone I 10 mg containing several analytical applications and all available standards 91625 Vascinone 10 mg and CRMs, listed in alphabetical order and ordered by plant genus. Please use the enclosed business reply 94882 (1S)-(−)-Verbenone 1 mL, 5 mL Phytochemical Standards card or our web portal 76418 Vescalagin 5 mg Analytical Standards and certified reference materials for constituents of medicinal plants (sigma-aldrich.com/lit-request) Table 1 New Analytical Standards of Active Ingredients and Marker to order the brochure. Substances for the Analysis of Herbal Medicinal Drugs

50560_Phytopharma Standards Brochure.indd 1 7/29/15 8:47 AM

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6 Analytix | 5: 2015

Marine Biotoxin CRMs Products from the National Research Council of Canada (NRC) Now Available through Sigma-Aldrich®

Matthias Nold, Product Manager Analytical Standards [email protected]

Several marine algae species are known to produce potent toxins hence the compounds need to be extracted and purified from the called phycotoxins. Through the food chain, these toxins can producing algae or contaminated seafood. accumulate and may end up in harmful concentrations in fish The National Research Council of Canada (NRC) has developed and marine invertebrates such as mussels, which can cause various certified reference materials (CRMs) for a wide range of regulated gastrointestinal and neurological illnesses when consumed by marine biotoxins. The suite of materials includes both pure calibration humans. solution CRMs and a selection of shellfish matrix CRMs. Following To reduce the risk of human intoxication, regulations have been extensive work on purification, the chemical structure and purity established with the aim of safeguarding seafood for human of the toxins are confirmed by a combination of NMR and LC-MS. consumption. The FDA has established action levels for saxitoxin, Concentration values are assigned by quantitative NMR and various brevetoxin-2, okadaic acid, domoic acid and azaspiracid equivalents chromatography techniques. Comprehensive homogeneity and as well as for ciguatoxins [1]. The European Commission defined stability tests are performed. limits for domoic acid, okadaic acid, dinophysistoxins, pectenotoxins, The range of biotoxin CRMs from NRC are now available through yessotoxins and azaspiracid equivalents in EC Regulation No. 853/2004 Sigma-Aldrich. The tables on the next page list the first series of and specified the detection method (preferably LC-MS/MS) in products that have been recently added to the Sigma-Aldrich Regulations No. 2074/2005 and No. 15/2011. portfolio. You can find these products on our website at Purified toxins for use as reference standards for analytical method sigma-aldrich.com/marinetoxins development and accurate quantification purposes are very difficult to obtain. The chemical structures are very complex (Figure 1), involving many stereogenic centers, macrocycles or fused ring systems. Organic synthesis is therefore very laborious when possible,

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FeatureStandards Article 7

OH CH CH H H 3 3 O CH3 O H C CH 3 H 3 O O O O OH O O O CH2 OH H3C O H O H O O O CH2 OH OH HO OH O O O O OH H3C O CH3 H3C O CH OH CH3 O O O 3 H C HO H 3 OH CH3 O CH CH H O 3 3 H3C O O OH Dinophysistoxin-1 (NRCCRMDTX1) Pectenotoxin-2 (NRCCRMPTX2B) H3C OH CH3 H3C OH CH2 Okadaic acid (NRCCRMOAC) H O CH CH2 O H 2 CH CH 3 NaO S O 3 H O H HN O H O O H O H O O H CH H CH H NaO S O H 3 3 O H H H O CH3 H O O O OH HO H O O O H O O H H H O O H O O H H3C OH H H H CH O CH3 OH 2 CH3 CH3 CH3 Yessotoxin (NRCCRMYTXB) Azaspiracid-3 (NRCCRMAZA3)

Figure 1 Chemical Structures of Some Regulated Marine Toxins

Cat. No. Description Composition* NRCCRMATX Anatoxin-a solution 30 μM in methanol: water ((9:91, v/v) with 0.01% acetic acid) NRCCRMAZA3 Azaspiracid-3 solution 1.3 μM in methanol NRCCRMCYN Cylindrospermopsin solution 30 μM in deionized water (filtered) NRCCRMDCNEOC Decarbamoylneosaxitoxin solution 30 μM in hydrochloric acid (3 mM) NRCCRMDCSTXB Decarbamoylsaxitoxin solution 65 μM in hydrochloric acid (3 mM) NRCCRMSPX1 13-Desmethylspirolide C solution 10 μM in methanol with 0.05% TFA NRCCRMDTX1 Dinophysistoxin-1 solution 19 μM in methanol NRCCRMDTX2 Dinophysistoxin-2 solution 10 μM in methanol NRCCRMGTX14C Gonyautoxin-1 and Gonyautoxin-4 solution GTX-1: 60 μM and GTX-4: 20 μM in hydrochloric acid (3 mM) NRCCRMGTX23C Gonyautoxin-2 and Gonyautoxin-3 solution Gonyautoxin-2: 114 μM and Gonyautoxin-3: 43 μM in hydrochloric acid (3 mM) NRCCRMHYTX 1-Homoyessotoxin solution 5 μM in methanol NRCCRMDMMCLR [Dha7]-Microcystin-LR solution 10 μM in methanol: water (1:1) (v/v) NRCCRMMCLR Microcystin-LR solution 10 μM in methanol: water (1:1) (v/v) NRCCRMMCRR Microcystin-RR solution 10 μM in methanol: water (1:1) (v/v) NRCCRMNEOC Neosaxitoxin solution 66 μM in hydrochloric acid (3mM) NRCCRMNODR Nodularin solution 12 μM in methanol: water (1:1) (v/v) NRCCRMOAC Okadaic acid solution 17 μM in methanol NRCCRMPTX2B Pectenotoxin-2 solution 5 μM in methanol NRCCRMSTXF Saxitoxin dihydrochloride solution 66 μM in hydrochloric acid (3mM) NRCCRMC12B N-Sulfocarbamoylgonyautoxin-2 and C1: 113 μM and C2: 34 μM in filtered, aqueous acetic acid (~17 μM, pH 5) N-Sulfocarbamoylgonyautoxin-3 solution NRCCRMYTXB Yessotoxin solution 5 μM in methanol Table 1 Marine Toxin Solution CRMs from NRC

Cat. No. Description Composition* NRCCRMZEROMUS Mussel tissue (Mytilus edulis) negative control NRCCRMAZAMUS Mussel tissue (Mytilus edulis) contaminated AZA1 – 1.16 μg/g, AZA2 – 0.273 μg/g and AZA3 – 0.211 μg/g with azaspiracids NRCCRMASPMUSD Mussel tissue (Mytilus edulis) contaminated Domoic acid: 48 μg/g, C5'-epi-Domoic acid: 1.9 μg/g, Isodomoic acid with domoic acid and isomers E: 1.0 μg/g (non-certified), Isodomoic acid D: 2.3 μg/g (non-certified), Isodomoic acid A: 1.5 μg/g (non-certified) Table 2 Marine Toxin Matrix CRMs from NRC * For the exact values including uncertainties, please refer to the certificate.

[1] http://www.fda.gov/downloads/Food/GuidanceRegulation/UCM252395.pdf

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8 Analytix | 5: 2015

Isomeric Distribution of Astaxanthin New Standards for All-Trans, 9-cis and 13-cis Astaxanthin

Xavier Àlvarez, Principal Scientist at Analytical Standards Division Greenaltech S.L. Like many other carotenoids, astaxanthin has several isomeric [email protected] structures depending on the configuration of the double bonds in Olga Durany, CEO Greenaltech the molecule. Its natural form in Haematococcus pluvialis is mainly [email protected] all-trans astaxanthin (over 90%) followed by 9-cis and 13-cis isomers Matthias Nold, Product Manager Analytical Standards (chemical structures shown in Figure 1), all of them stabilized [email protected] as fatty acid esters [1, 2]. Abundance of the cis isomers increases Sigma-Aldrich® recently when all-trans astaxanthin is manipulated in quantitative analytical introduced analytical methods (mainly during the hydrolysis to the free form), during the standards for the most extraction process to nutraceutical supplements, or due to exposure abundant isomers of to light, high temperatures or organic solvents [3, 4]. The isomeric astaxanthin: all-trans, 9-cis ratio of astaxanthin in Haematococcus pluvialis could be an indicative and 13-cis. Astaxanthin of biomass processing or storage. Continuous research is being is a red, fat-soluble conducted to obtain alternative sources of natural astaxanthin, and pigment from the group recent studies have shown that astaxanthin of microalgal origin of xanthophylls, naturally has a different isomeric profile compared to astaxanthin from other occurring in various microalgae and yeast species. Interest in this sources [3]. Moreover, cis isomers and especially 9-cis-astaxanthin carotenoid from the pharmaceutical and nutraceutical industries have been reported to exhibit higher antioxidant activity than the has been increasing exponentially during the last decade due to its all-trans form [5]. Therefore, it is important to be able to accurately health benefits in the human diet, such as cardiovascular disease and identify and quantify not only all-trans-astaxanthin, but also its 9-cis cancer prevention, anti-inflammation, antidiabetic and antioxidant and 13-cis isomers. effects [1]. One of the main natural sources of astaxanthin is the In the past, due to the lack of suitable standards, 9-cis and 13-cis microalgae Haematococcus pluvialis, which can accumulate over 2% astaxanthin isomers had been analyzed and quantified by HPLC-PDA of this pigment in the dried biomass. This microalgae is the primary with all-trans-astaxanthin as external standard, applying different synthesizer of astaxanthin in the marine trophic chain, responsible for correction factors to compensate for the different isomeric response the characteristic pink coloration of salmon, shrimp and many other factors [6–8]. This approach may lead to certain errors when organisms [2]. chromatographic conditions vary from the referenced method. For this reason, the objective of this application note is to develop a quantification method for all-trans, 9-cis and 13-cis-astaxanthin O using isomerically pure standards, which have recently become H C OH CH CH 3 H3C CH3 3 3 commercially available from Sigma-Aldrich.

CH3 CH3 H3C CH3 HO CH3 Experimental O all-trans-astaxanthin OH Sample Preparation O Haematococcus pluvialis extract was prepared for chromatographic H3C CH3 O H3C analysis as described in the literature. [8] In brief, fine powdered H3C OH CH3 biomass was extracted three times with acetone by sonication and H3C mixed with all-trans-β-Apo-8’-carotenal as internal standard (Sigma H C CH CH3 CH3 3 3 10829), whereas oleoresins were directly dissolved in acetone and 9-cis-astaxanthin mixed with the internal standard. Astaxanthin esters were hydrolyzed OH with cholesterol esterase and extracted with petroleum ether. The O sample was finally evaporated and reconstituted in acetone prior to H3C CH3 H3C HPLC analysis.

H3C O HPLC Conditions H3C OH 10 µl of sample were injected into a YMC-carotenoid column

H3C (4.6 x 250 mm, 5 µm) at 1 ml/min flow. The mobile phase program H C CH CH3 CH3 3 3 is described in Table 1. UV/VIS detection was carried out by 13-cis-astaxanthin photodiode array at 474 nm.

Figure 1 Most Abundant Natural Geometric Isomers of Astaxanthin

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FeatureStandards Article 9

Results and Discussion Chromatographic separation of an equimolar mixture of all-trans, 9-cis and 13-cis-astaxanthin (10 ppm each) is presented in Figure 2. As can be observed, at an equal concentration, all-trans-astaxanthin showed higher molar absorptivity at 474 nm, followed by the 9-cis and 13-cis isomers. This is why different correction factors have to be applied when cis isomers are quantified using all-trans astaxanthin as a standard. These factors have to be adjusted for any given mobile phase program, since solvation with different organic solvents at the moment of detection changes their molar absorptivity. Results also depend on the detection wavelength, because each isomer has a different maximum absorbance. Figure 3 and Figure 4 show the calibration curves obtained for the quantification of astaxanthin isomers with the three pure isomeric standards, and with using all-trans astaxanthin to quantify 9-cis and 13-cis isomers (applying correction factors of 1.1 and 1.3, respectively) [6–8]. The quantification method based on the correction factors clearly Figure 2 Chromatographic Separation of 13-cis, All-Trans and 9-cis underestimates the calculated concentrations. For the 9-cis isomer, Astaxanthin Isomers (Equimolar Mixture) the results were about 35% lower than the value determined with the specific standard, whereas for 13-cis-astaxanthin, the calculated concentrations were underestimated by around 45%. This proves that quantification of cis isomers using all-trans-astaxanthin can only be used when correction factors are experimentally adjusted for each chromatographic condition, and this can only be carried out using pure standards of the 9-cis and 13-cis isomers. Otherwise, results could be highly biased, depending on the proportion of the cis isomers in the product. Alternatively, isomerically pure 9-cis and 13-cis-astaxanthin can be used for an accurate external standard quantification of astaxanthin isomers independently from the chromatographic conditions. This would be the best option for the analysis of more complex real life samples from different origins, where the presence of other carotenoids may need different chromatographic conditions to avoid interferences (Figure 5). These two quantification methods were employed to determine Figure 3 Calibration Curves of 9-cis-astaxanthin Using Quantification Meth- the concentration of all-trans, 9-cis and 13-cis-astaxanthin in four ods Based on Correction Factors and the Pure Standard Haematococcus pluvialis dried biomass samples and four oleoresins extracted from the same microalgae. The results are summarized in Table 2, whereas Figure 6 compares the relative astaxanthin isomeric contribution obtained with the two different quantification methods. Total astaxanthin was underestimated by around 10% when the quantification by factors was employed. This bias is lower than that determined for 9-cis and 13-cis-astaxanthin separately, because all- trans is the most abundant isomer in Haematococcus pluvialis. These differences were even more apparent when isomeric contributions were compared, where 9-cis and 13-cis-astaxanthin were clearly underestimated using the correction factors. It is notable that the average contribution of the all-trans isomer in biomass is higher than its value in oleoresins. This can be explained by taking into account that oleoresins have undergone a process of supercritical extraction from the biomass, and all-trans can isomerize to cis-astaxanthin during this step. This highlights the importance of Figure 4 Calibration Curves of 13-cis-astaxanthin Using Quantification being able to quantify 9-cis and 13-cis-astaxanthin accurately using Methods Based on Correction Factors and the Pure Standard pure isomeric standards.

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10 Analytix | 5: 2015

Figure 5 Haematococcus Pluvialis Oleoresin Chromatogram Figure 6 Astaxanthin Isomeric Contributions in Oleoresins (OR) and Haematococcus Pluvialis Biomass Quantified with Standards (Std) and Correction Factors (Fac)

Time (min) Methanol t-butyl-methyl 1% H PO 3 4 Cat. No. Description Purity (HPLC) Package Size ether aqueous 41659 all-trans-Astaxanthin ≥97% 1 mg, 5 mg 0 81 15 4 51881 9-cis-astaxanthin ≥90% 1 mg 15 66 30 4 52991 13-cis-astaxanthin ≥90% 1 mg 23 16 80 4 7168 all-trans-Lutein ≥97% 1 mg, 5 mg 27 16 80 4 Table 3 Analytical Standards of Astaxanthin Isomers and All-Trans-Lutein 27.1 81 15 4 35 81 15 4

Table 1 HPLC Mobile Phase Program

mg astaxanthin/g Quantified by Standards mg astaxanthin/g Quantified by Factors all-trans 9-cis 13-cis total all-trans 9-cis 13-cis total OR1 57.3 7.97 11.9 77.2 57.3 4.91 6.46 68.70 OR2 62.1 8.65 12.4 83.1 62.1 5.49 6.84 74.44 OR3 65.3 10.8 15.5 91.6 65.3 6.96 8.69 80.94 OR4 69.3 11.5 15.5 96.3 69.3 7.46 8.70 85.42 BM1 17.9 1.40 1.84 21.2 17.9 0.79 0.92 19.65 BM2 21.1 1.58 2.20 24.8 21.1 0.93 1.15 23.15 BM3 18.9 2.18 1.61 22.7 18.9 1.37 0.79 21.07 BM4 18.9 2.15 1.67 22.7 18.9 1.35 0.83 21.06

Table 2 Astaxanthin Quantification in Oleoresins (OR) and Haematococcus Pluvialis Biomass

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FeatureStandards Article 11

Conclusions References An accurate quantification of all-trans, 9-cis and 13-cis-astaxanthin [1] Ambati, R. R.; Phang, S. M.; Ravi, S.; Aswathanarayana, R. G. Astaxanthin: can be achieved when recently commercially available pure Sources, extraction, stability, biological activities and its commercial standards are used for each isomer. applications – a review. Mar. Drugs, 2014, 12, 128-152. [2] Higuera-Cipara, L.; Félix –Valenzuela, L; Goycoolea, F.M. Astaxanthin: Alternatively, quantification of these isomers can be carried out with A review of its chemistry and applications. Crit. Rev. Food Sci. Nutr., 2006, 46, all-trans-astaxanthin employing correction factors for the 9-cis and 1040-8398. 13-cis isomers. Nevertheless, these factors have to be empirically [3] Lin, W. C.; Chien, J. T.; Chen, B.H., Determination of carotenoids in spear adjusted for each chromatographic condition using the isomerically shrimp shells (Parapenaeopsis hardwickii) by liquid chromatography. J. Agric. pure standards, otherwise the results can be highly biased. Food Chem, 2005, 53, (13): 5144-5149. [4] Yuan, J. P.; Chen, F. Isomerization of trans-astaxanthin to cis-isomers in organic All analytical standards used for this study are available from Sigma- solvents. J. Agric. Food Chem, 1999, 47, 3656-3660. Aldrich (Table 3). A complete listing of all carotenoid standards can [5] Liu, X.; Osawa, T. Cis astaxanthin and especially 9-cis astaxanthin exhibits a be found on sigma-aldrich.com/carotenoids higher antioxidant activity in vitro compared to all-trans isomer. Biochem. Biophys. Res. Commun., 2007, 357, 187-193. [6] Rui Chen, J. R. Quantitative analysis of astaxanthin in dietary supplements by ultraperformance convergence chromatography (UPC2). Waters application note APNT134820709. [7] Sun, W. H.; Leng, K. L.; Xing, L. H.; Zhu, M.; Zhai, Y. X.; Miao, J. K.; Zhu, L. L., Determination of astaxanthin in antarctic krill oil by GPC-high performance liquid chromatography and calculation of correction factor. Chi. J. Anal. Lab., 2013, 2, 26-30. [8] Astaxanthin content in AstaREAL® L10, V1 May 2009. http://www.fujihealthscience.com/Assay-Method_AstaREAL-L10.pdf

NEW FROM SIGMA-ALDRICH® Certified Reference Materials (CRMs) For the Petrochemical Industry

From ISO/IEC 17025 and ISO Guide 34. Accredited leading producer, Paragon Scientific. The Paragon range includes more than 600 products with a wide variety of Petrochemical Certified Reference Materials. For more information, visit sigma-aldrich.com/paragon

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12 Analytix | 5: 2015

Traceability Means Reliability New Organic TraceCERT® CRMs for Chromatography

Matthias Nold, Product Manager Analytical Standards [email protected] Alexander Rück, Supervisor R&D [email protected] Christine Hellriegel, Senior Scientist R&D [email protected] There are many factors that determine the quality of an analytical measurement. Reliable analytical methods, sophisticated instruments and modern laboratory equipment are as important as the proficient skills and experience of the scientists who perform the measurements. However, even if all these requirements are fulfilled, the quality of analytical results still stands and falls with the quality of the reference materials used to calibrate the analytical instruments. This fact is taken into account by ISO/IEC17025 which advises (if applicable) the use of SI traceable certified reference materials for this purpose. Traceability can be achieved by measuring against a primary reference material from a national metrological institute (such as NIST). However, the number of organic compounds is virtually endless, so finding an appropriate metrological standard might prove difficult to impossible for most organic analytes. This can be resolved by using a relative primary analytical method the results of which are independent of the chemical structure. A very efficient method which fulfills this requirement is quantitative NMR (qNMR)[1-3]. It is essentially applicable for all organic compounds, and since the value of the integrals of the proton signals is not affected by the chemical structure, it allows quantitative comparison between different organic molecules. Hence, a limited set of NIST SRM-traceable internal standards is sufficient to establish traceability for any organic molecule. Sigma-Aldrich® Buchs (Switzerland) has more than six years of experience in manufacturing CRMs using qNMR under ISO/IEC17025 We continually work on the expansion of this portfolio. The most and ISO Guide 34 double accreditation. This is the highest possible recent additions are shown below. These include glyphosate, one of accreditation that can be achieved by a reference material producer. the most important herbicides worldwide, linalool, a terpene alcohol Using this approach, the portfolio of neat organic TraceCERT CRMs which is widely used as a fragrance in personal hygiene products or has been continually expanded and currently consists of nearly Tris(2-chloroethyl) phosphate which finds use as a flame retardant 200 products. and plasticizer in polymers. The products can be found online at The organic TraceCERT certified reference materials are characterized sigma-aldrich.com/organiccrm where you can find a complete and by: up-to-date list of all products and download example certificates and technical articles. • Certified content by quantitative NMR (qNMR) • Compliance to ISO/IEC 17025 and ISO Guide 34 Cat. No. Description Package Size • Superior level of accuracy, calculated uncertainties and 18191 2,4-Dinitrotoluene 100 mg lot-specific values 13295 Ethyl benzenesulfonate 120 mg Traceability to NIST SRM 89432 Glyphosate 50 mg • 61706 Linalool 50 mg Comprehensive documentation delivered with the product • 61927 Mesitylene 100 mg (certification according to ISO Guide 31) 53706 Methyl butyrate 100 mg The product range includes a wide variety of important analytical 96382 Tris(2-chloroethyl) phosphate 100 mg analytes for food and environmental testing such as pesticides, PAHs, amino acids, plasticizers, etc. sigma-aldrich.com/analytixsigma-aldrich.com/organiccrm 12 Back to Table of Contents

FeatureStandards Article 13

CH3 O O H H3C NO2 N CH HO P OH 2 CH3 HO CH3 OH NO2 2,4-Dinitrotoluene Glyphosate Linalool

CH 3 O O Cl Cl OPO H3C OCH3 O H3C CH3 Cl Mesitylene Methyl butyrate Tris(2-chloroethyl) phosphate

References: [2] Weber M.; Hellriegel C.; Rueck A.; Sauermoser R.; Wuethrich J., Using [1] Weber M.; Hellriegel C.; Rueck A.; Wuethrich J.; Jenks P., Using high highperformance quantitative NMR (HP-qNMR®) for certifying traceable and performance 1H NMR (HP-qNMR®) for the certification of organic reference highly accurate purity values of organic reference materials with uncertainties materials under accreditation guidelines – Describing the overall process <0.1%, Accred. Qual. Assur. 18, 2013, 91-98. with focus on homogeneity and stability assessment, JPBA 93, [3] Weber M.; Hellriegel C.; Rueck A.; Wuethrich J.; Jenks P.; Obkircher M., Method 2014, 102-110. development in quantitative NMR towards metrologically traceable organic certified reference materials used as 31P qNMR standards, Anal Bioanal Chem., 2015, 407: 3115-3123.

Standards for Personal Care and Cosmetic Products For Beauty with Safety

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14 Analytix | 5: 2015

Glucuronide Drug Testing Applications by Mass Spectrometry Certified Spiking Solutions® of Glucuronides

Maximilian Magana, Technical Marketing Specialist Cerilliant® offers a wide selection of solution-based Certified [email protected] Reference Materials (CRMs) for native and stable-labeled glucuronides Derrell Johnson, Manager, New Product Strategy & Tactical Marketing of numerous drug classes including alcohol/ethanol, anesthetics, [email protected] benzodiazepines, cannabinoids, immunosuppressants, opiates, The process of glucuronidation involves the enzyme-mediated transfer steroids/hormones, and synthetic analgesics. Our glucuronide of glucuronic acid to an exogenous substance during liver metabolism. CRMs are packaged in a USDEA and Health Canada (with TK #s)- Many pharmaceuticals and illicit drugs of abuse are excreted from exempt solution format for added convenience. the human body as glucuronide conjugates. Glucuronides possess Register at sigma-aldrich.com/registercerilliant to receive news on many properties that make them desirable for clinical, forensic, sports the latest reference standards introduced by Cerilliant. doping and drug development testing applications, such as their high View sigma-aldrich.com/cerilliant to learn more about Cerilliant’s concentration in urine, a longer half-life compared to an unconjugated extensive catalog offering of CRMs and Certified Spiking Solutions. metabolite, and their presence in numerous bodily fluids [1]. Over the last decade, methods for the analysis of glucuronides have References: been developed to improve existing drug testing strategies. One such [1] Kintz, P. Toxicological Aspects Of Drug-Facilitated Crimes. Burlington: example involves ethyl glucuronide (EtG), a urinary metabolite of Elsevier Science, 2014. Print. alcohol. EtG offers several advantages over ethanol in alcohol testing [2] DasGupta, A. Alcohol and its biomarkers: Clinical aspects and laboratory determination. San Diego: Elsevier, 2015. Print. including a longer detection window, greater accuracy and sensitivity, [3] Cao, Z.; Kaleta E.; and Wang P., Simultaneous Quantitation Of 78 Drugs and detectability in alternate matrices such as hair and meconium. As a And Metabolites In Urine With A Dilute-And-Shoot LC-MS-MS Assay. result of these advantages, EtG has become a widely used biomarker in J Anal Toxicol 39.5, 2015: 335-346. Web. 7 Aug. 2015. alcohol testing not just for medico-legal and forensic investigations but [4] Morris, A. A. et al., Rapid Enzymatic Hydrolysis Using A Novel also for clinical trials [1], [2]. Recombinant – Glucuronidase In Benzodiazepine Urinalysis. Historically, glucuronides have been analyzed using gas J Anal Toxicol 38.8, 2014: 610-614. Web. 7 Aug. 2015. chromatography-mass spectrometry (GC-MS) methods. [5] Xiong, Lingjuan et al., Determination Of Co-Administrated Opioids And Benzodiazepines In Urine Using Column-Switching Solid-Phase Despite their historical use, GC-MS glucuronide methods are time- Extraction And Liquid Chromatography–Tandem Mass Spectrometry. consuming and in many cases require sample pre-treatment steps. J Chromatogr A 1395, 2015: 99-108. Web. 7 Aug. 2015. These steps include enzymatic (β-glucuronidase) or acid hydrolysis [6] Toennes, Stefan W. et al., Pitfall In Cannabinoid Analysis – Detection to cleave the glucuronide and free the parent drug followed by Of A Previously Unrecognized Interfering Compound In Human Serum. derivatization for analysis [2], [3]. Anal Bioanal Chem 407.2, 2014: 463-470. Web. 7 Aug. 2015. The efficiency of enzymatic or acid hydrolysis is subject to variables such as enzyme or acid concentration and incubation time. If these variables are not properly evaluated and optimized during method development, method accuracy can be impacted. Acid hydrolysis of the benzodiazepine metabolites oxazepam and temazepam glucuronide, for example, induces degradation of the conjugates into benzophenones [3], [4]. With liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, unlike those by GC-MS, the analyst is able to directly measure glucuronide concentrations without sample hydrolysis and derivatization, although the hydrolysis step can continue to be used if desired [5], [6]. LC-MS/MS methods also allow the simultaneous determination of multiple, small volume analytes in a single run – a major benefit to laboratories that need to determine co-administrated medications and their glucuronide metabolites in a biological matrix [5], [6]. Owing to its high sensitivity for small sample volumes and fewer sample preparation steps, LC-MS/MS has increasingly replaced GC-MS as the method of choice in some forensic and clinical toxicology glucuronide testing applications [6].

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FeatureStandards Article 15

Glucuronide Certified Spiking Solutions Cat. No. Brand Description Package Size Alcohol/Ethanol E-015 Cerilliant Ethyl-β-D-glucuronide 1.0 mg/mL in Methanol E-016 Cerilliant Ethyl-β-D-glucuronide 100 µg/mL in Methanol

E-048 Cerilliant Ethyl-β-D-glucuronide-D5 100 µg/mL in Methanol

E-063 Cerilliant Ethyl-β-D-glucuronide-D5 1.0 mg/mL in Methanol Anesthetics P-082 Cerilliant Propofol β-D-glucuronide sodium salt 100 µg/mL (as free acid) in Methanol Benzodiazepines L-021 Cerilliant Lorazepam glucuronide 100 µg/mL in 50:50 Acetonitrile:Water O-023 Cerilliant Oxazepam glucuronide 100 µg/mL in Methanol

O-038 Cerilliant Oxazepam-D5 glucuronide 100 µg/mL in Methanol T-050 Cerilliant Temazepam glucuronide lithium salt 100 µg/mL (as free acid) in Methanol Cannabinoids T-038 Cerilliant (+)-11-Nor-Δ9 -THC-9-carboxylic acid glucuronide (Not to be used for immunoassay) 100 µg/mL in Methanol 9 T-080 Cerilliant (±)-cis-11-Nor-9-carboxy-Δ -THC-D3 glucuronide 100 µg/mL in Methanol Immunosuppressants M-135 Cerilliant Mycophenolic acid-β-D-glucuronide 1.0 mg/mL in Acetonitrile Opiates B-035 Cerilliant Buprenorphine-3-β-D-glucuronide 100 µg/mL in Methanol

B-060 Cerilliant Buprenorphine glucuronide-D4 100 µg/mL in Methanol C-087 Cerilliant Codeine-6-β-D-glucuronide 100 µg/mL in Water:Methanol (80:20) C-126 Cerilliant Codeine-6-β-D-glucuronide 1.0 mg/mL in Water:Methanol (80:20)

C-138 Cerilliant Codeine-6-β-D-glucuronide-D3 100 µg/mL in Water:Methanol (80:20) D-108 Cerilliant Dihydromorphine-3-β-D-glucuronide 1.0 mg/mL in 1:1 Methanol:Water D-109 Cerilliant Dihydrocodeine-6-β-D-glucuronide 1.0 mg/mL in 1:1 Methanol:Water H-051 Cerilliant Hydromorphone-3-β-D-glucuronide 100 µg/mL in Methanol:Water(1:1)

M-017 Cerilliant Morphine-3-β-D-glucuronide-D3 100 µg/mL in Methanol w/0.05% NaOH M-018 Cerilliant Morphine-3-β-D-glucuronide 100 µg/mL in 1:1 Methanol:Water M-031 Cerilliant Morphine-3-β-D-glucuronide 1.0 mg/mL in Methanol w/0.05% NaOH M-046 Cerilliant Morphine-6-β-D-glucuronide 1.0 mg/mL in Water:Methanol (80:20) M-096 Cerilliant Morphine-6-β-D-glucuronide 100 µg/mL in Methanol:Water(1:1)

M-120 Cerilliant Morphine-6-β-D-glucuronide-D3 100 µg/mL in Methanol:Water(1:1) N-045 Cerilliant Norbuprenorphine glucuronide 100 µg/mL in Methanol

N-097 Cerilliant Norbuprenorphine glucuronide-D3 100 µg/mL in Methanol N-099 Cerilliant Naloxone-3-β-D-glucuronide 1.0 mg/mL in 10% Water in Methanol N-104 Cerilliant 6β-Naltrexol-3-β-D-glucuronide 1.0 mg/mL in 20% Water in Acetonitrile O-030 Cerilliant Oxymorphone-3-β-D-glucuronide 100 µg/mL in 1:1 Methanol:Water

O-031 Cerilliant Oxymorphone-D3-3-β-D-glucuronide 100 µg/mL in 1:1 Methanol:Water Steroids/Hormones

A-099 Cerilliant Androsterone-D4 glucuronide 100 µg/mL in Methanol Synthetic Analgesics T-060 Cerilliant Tapentadol-β-D-glucuronide 100 µg/mL in 1:1 Acetonitrile:Water

T-067 Cerilliant Tapentadol-D3-β-D-glucuronide 100 µg/mL in 1:1 Acetonitrile:Water

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16 Analytix | 5: 2015

New Hellma® TrayCell™ – Photometric Micro Volume Analysis in the Nanoliter Range DNA/RNA – Simply Analyzed

Magdalena Ulman, Product Manager Analytical Reagents of the optical window. The cap with mirror provides a well-defined [email protected] optical light path and prevents the sample from drying up. The The TrayCell is a fiber-optic ultra-micro cell designed for the UV/Vis measurement remains reproducible because the sample will not micro volume analysis of DNA/RNA and proteins, as it enables highly be enriched by evaporation of the solvent. During filling and accurate analysis of extremely small samples (measurement volume: cleaning stages, the cell remains in the photometer. This guarantees 0.7 to 10 µL) with remarkable reproducibility. The dimensions of the a continuously identical position of the aperture in the light beam TrayCell are equivalent to a standard cuvette in order to work in most and no variation in comparison to the reference measurement. spectrophotometers. Using the 1 mm or 0.2 mm cap creates a defined optical light path of Typical applications of the TrayCell are as follows: 1 mm and 0.2 mm respectively. This generates virtual dilution factors of 1:10 or 1:50 in comparison to a measurement with a standard Nucleic acid analysis • 10 mm cuvette. This feature saves time and avoids dilution errors. If • Determination of the incorporation frequency of fluorescent dye desired, samples can be retrieved after the measurement for further labels (FOI) processing. The required sample volume for the 1 mm cap is 3 μl to • Protein analysis (A280, BCA, Lowry, etc.) 5 μl and for the 0.2 mm cap 0.7 μl to 4 μl. All UV/Vis analysis utilizing the wavelength range of 190–1100 nm With the Hellma TrayCell, the average dynamic range for dsDNA • is between 2 ng/μl and 5000 ng/μl. The average dynamic range Due to the integrated beam deflection and the use of fiber-optic depends strongly on the type of photometer used. cables, it is possible to measure the sample directly on the surface

Cat. No. Description Window Material Width x Depth [mm] Height [mm]; Center Height [mm] Volumes [µL] Path Length [± 0.02 mm] Z802573 TrayCell Quartz SUPRASIL® 12.5 x 12.5 68.5; 8.5 0.7–10 0.2 (factor 50) or 1 (factor 10) 75; 15 80; 20 Z802697 TrayCell Quartz SUPRASIL 12.5 x 12.5 53; 8.5 0.7–10 0.2 (factor 50) or 1 (factor 10) 59.5; 15 64.5; 20 Material Path Length Volumes [μl] [± 0.02 mm] Z802808 TrayCell cap Stainless steel cap 1.0 (factor* 10) 3–5 Z802905 TrayCell cap with integrated 0.2 (factor* 50) 0.7–4 Z803014 TrayCell cap mirror (Quartz glass 2.0 (factor* 5) 6 –10 SUPRASIL with Z803138 TrayCell cap aluminum mirror 0.1 (factor* 100) 0.7–3 coating) Table 1 TrayCell and Caps. Please Visit our Website for More Information at sigma-aldrich.com/hellma * Factor – dilution factor compared to a standard cell with a path length of 10 mm sigma-aldrich.com/analytixsigma-aldrich.com/hellma 16 Back to Table of Contents

FeatureLabware Article 17

Timestrip® The Smart Way to Measure Time and Temperature

Daniel Weibel, Product Manager Analytical Reagents Successful Timestrip applications include filters of all types (air, fridge, [email protected] freezer, cooker hood, water, vacuum); compressed gas or ink jet In this fifth article about Timestrip (Analytix issues 1-4 2015) we cartridges, industrial and vehicle parts, cosmetics and personal care explore the versatility of Timestrip time lapse indicators. products such as toothbrushes and cosmetics, and healthcare and medical devices such as disposable hospital curtains, nebulizer cups and IV sets.

Diagnostic Testing – Minutes, Hours or Days Timestrip Timestrip can be made for shorter time periods, which are ideal for medical devices like IVs, which need changing after three days, or diagnostic testing kits. ROM Plus is a self-contained test designed They can be used in a laboratory environment for monitoring expiry by Clinical Innovations that comes packaged for use in either a POC dates of samples, chemicals and solutions, but they can also be (point-of-care) setting or in a lab. The cassette has a built-in 20 minute integrated into parts and devices designed or manufactured by your Timestrip indicator, a lateral flow test strip and a patient ID field organization. The case studies explored here are for illustration only (Figure 2). The results manifest within 5–20 minutes, so 5, 10 and and the devices and customized indicators pictured cannot be 20 minutes are clearly marked on the Timestrip, allowing clinicians bought from Sigma-Aldrich® or Timestrip. If you have a similar to have a clear visual indication of when results are ready. With an application that could benefit from integrating a Timestrip time accurate diagnosis of PROM (premature rupture of membranes), reminder, please contact [email protected] for more information patients can receive interventions to minimize the on stock product or fully customized options. You can customize the risk of complications. graphics, the shape and the time period of a Timestrip label. For more information on Timestrip, visit our website at Replacing Parts – Months and Years Timestrip sigma-aldrich.com/timestrip or Timestrip reminders are used extensively with filters and other contact [email protected] replaceable parts with a recommended or regulated ‘life in service’. Providing a Timestrip with the part encourages the user to replace the part on time and buy a replacement. Timely replacement not only drives up the frequency of purchase, but by improving device performance it can improve customer satisfaction and loyalty. Timestrip can be stuck onto a part or filter or inserted into molded housings (Figure 1). Figure 2

Cat. No. Brand Description 93064 Sigma-Aldrich® Timestrip 1 month 07603 Sigma-Aldrich Timestrip 3 months 06797 Sigma-Aldrich Timestrip 6 months 74831 Sigma-Aldrich Timestrip 12 months 03849 Sigma-Aldrich Timestrip 3 months, key chain 06929 Sigma-Aldrich Timestrip 12 months, key chain 06693 Sigma-Aldrich Timestrip Plus -20 °C 92210 Sigma-Aldrich Timestrip Plus 0 °C 08168 Sigma-Aldrich Timestrip Plus 8 °C 92451 Sigma-Aldrich Timestrip Plus 25 °C 80474 Sigma-Aldrich Timestrip Plus 30 °C 80476 Sigma-Aldrich Timestrip Plus Duo 10 °C and 34 °C Figure 1 Table 1 Timestrip Indicators are Available in Package Sizes of 10 EA, 100 EA and 500 EA

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18 Analytix | 5: 2015

A Safer Way to Open Glass Ampoules SnapITs are easy-to-use, multi-use ampoule openers that prevent the possibility of injury sometimes seen when ampoules are opened by hand.

1 2 3

Ampoule Size Cat. No. Brand Description Compatibility 94791 Sigma- Ampoule opener – Regular 1–2 ml, 5–10 ml, Aldrich® 10 –15 ml 43917 Sigma- Ampoule opener – Large 5–10 ml, 10 –15 ml, 1 INSERT the ampoule lid into your multi-use SnapIT Aldrich 20–25 ml 2 SNAP open the ampoule 3 EJECT the ampoule lid directly into a sharps container SnapIT ampoule openers help you move toward a safer workplace. They are available in two slimline cylindrical designs: regular and large.

New TLC Staining Solution for Boronic Acids Curcumin solution 28982 effectively stains various types of boron-containing species, such as: • Free boronic acids • Boronic acid pinacol esters Potassium trifluoroborate salts • 1 691615 (+)-Vinylboronic acid pinanediol ester For rapid monitoring of your Suzuki-Miyaura cross-coupling, or any other organic reaction 2 674710 3-Methoxy-1-propyn-1-ylboronic acid involving boronic acids and related derivatives, try curcumin solution 28982. pinacol ester For more information on TLC derivatization, visit sigma-aldrich.com/derivatization 3 346225 (2-Methylpropyl)-boronic acid 4 683590 Potassium trans-3-methoxy- 1-propenyltri-fluoroborate [1] Lawrence, K.; Flower, S. E.; Kociok-Kohn, G.; Frost, C. G.; James, T. D., A simple and effective colorimetric technique for the detection of boronic acids and their derivatives. Anal. Methods 2012, 4, 2215–2217.

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ChromatographyFeature Article 19

Reversed-phase HPLC Buffers High-quality Buffers (Solutions, Solids or Concentrates)

Shyam Verma, Market Segment Manager Buffer’s effect on detection: The choice of buffer is also dependent [email protected] upon means of detection. For traditional UV detection, the buffer Consideration of the effects of pH on analyte retention, type of buffer needs to be effectively transparent in this region especially, critical to use and its concentration, solubility in the organic modifier and its for gradient separations. Buffers listed in Table 1 have low enough effect on detection are important in reversed-phase chromatography absorption below 220 nm. (RPC) method development of ionic analytes. An improper choice Phosphoric acid and its sodium or potassium salts are the most of buffer, in terms of buffering species, ionic strength and pH, can common buffer systems for reversed-phase HPLC. Phosphonate result in poor or irreproducible retention and tailing in reverse-phase buffers can be replaced with sulfonate buffers when analyzing separation of polar and ionizable compounds. organophosphate compounds. With the growth in popularity of Problems, such as partial ionization of the analyte and strong LC-MS, volatile buffer systems, such as TFA, acetate, formate and interaction between analytes and residual silanoles or other active ammonia, are frequently used due to compatibility with mass sites on the stationary phases, can be overcome by proper mobile spectral (MS) detection. In regard to the issue of suppression of phase buffering (maintaining the pH within a narrow range) and ionization, formate and acetate are ideal choices for positive-ion choosing the right ionic species and its concentration (ionic strength) mode detection. TFA, however, can negatively impact detector in the mobile phase [1–2]. In sensitive LC-MS separations that depend response even in positive-ion mode [4, 5], while it strongly suppresses heavily on the correct choice of acid, base, buffering species and ionization with negative ion mode. Acetic acid is good for negative- other additives [3], a buffer must be chosen based on its ability to ion mode. LC-MS applications further limit buffer selection and maintain, and not suppress, analyte ionization in the MS interface. buffer concentration. Buffer Selection References: [1] McMaster, M.C. HPLC A Practical User’s Guide, VCH Publishers, Inc.: New York, The typical pH range for reversed-phase-on-silica-based packing is NY, 1994; 85. pH 2 to 8. Choice of buffer is typically governed by the desired pH. [2] Poole, C.F. and Poole, S.K., Chromatography Today, Elsevier Science: It is important that the buffer has a pKa close to the desired pH since Amsterdam, Netherlands, 1991; 431. buffers control pH best at their pKa. A rule of thumb is to choose a [3] Analytix, Five-part series on Mobile Phase Additives for LC-MS, Issue 3, 2006 buffer with a pKa value <2 units of the desired mobile phase pH (sigma-aldrich.com/analytix). (see Table 1). [4] Temesi, D.; Law, B., The Effect of LC Eluent Composition on MS Response Using Electrospray Ionization, LC-GC, 1999, 17:626. Buffer pKa (25 °C) Useful pH Range [5] Apffel, A. et. al., Enhanced Sensitivity for Peptide Mapping with Electrospray TFA 0.5 <1.5 Liquid Chromatography-Mass Spectrometry in the Presence of Signal Sulfonate 1.8 <1–2.8 Suppression Due to Trifluoroacetic Acid-Containing Mobile Phases, J. Chrom. Phosphate 2.1 1.1–3.1 A., 1995, 712:177. Chloroacetate 2.9 1.9–3.9 Formate 3.8 2.8–4.8 HPLC-grade Buffers and Additives from Acetate 4.8 3.8–5.8 Sigma-Aldrich/Fluka® Sulfonate 6.9 5.9–7.9 Cat. No. Description Package Size Phosphate 7.2 6.2–8.2 17836 Ammonium acetate 50 g, 250 g Ammonia 9.2 8.2–10.2 17843 Ammonium formate 50 g, 250 g Phosphate 12.3 11.3–13.3 17837 Ammonium hydroxide solution in water 100 mL, 1 L Table 1 HPLC Buffers, pKa Values and Useful pH Range 17842 Ammonium phosphate monobasic 250 g 17839 Ammonium trifluoroacetate 10 g, 50 g Buffer concentration: Generally, a buffer concentration of 10–50 mM 17835 Potassium phosphate dibasic anhydrous 250 g is adequate for small molecules. 17841 Sodium formate 50 g, 250 g Buffer solubility: A general rule is no more than 50% organic 71633 Sodium phosphate dibasic dehydrate 250 g should be used with a buffer. This will depend on the specific buffer 17844 Sodium phosphate monobasic anhydrous 50 g, 250 g as well as its concentration. 17840 Sodium trifluoroacetate 10 g 09746 Trifluoroacetic acid:Triethylamine (2M:1M) 500 mL 09747 Trifluoroacetic acid:Triethylamine (2M:2M) 100 mL

For a complete list of HPLC buffers and additives, please refer to our online product catalog sigma-aldrich.com/hplc

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20 Analytix | 5: 2015

Microbiology has Moved into Microbiology Focus If you are interested in microbiology, we recommend you register for Microbiology Focus

• News about microbiology • Validations • Relevant articles from the field • Education • Valuable technical literature and references • Fun • Applications • And a lot more …

Would you like to register? Please visit sigma-aldrich.com/mibifocus

Your Resource to Find the Best Solvents for Your Application

Our Solvent Center page (sigma-aldrich.com/solvents) can quickly guide you to the solvent or grade best suited to support your application: • Browse our online catalog by solvent, grade or market and application • Download helpful brochures and flyers • Access further information on packaging options and returnables • Find out what special offers are available You can explore all that and more at sigma-aldrich.com/solvents

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FeatureTitration Article 21

Water Determination in Cosmetic Products Karl Fischer Titration with Hydranal® Reagents

Andrea Ziegler, Market Segment Manager listed them below (Table 1). Both the one-component Hydranal [email protected] Composite 5 or the two-component Hydranal Titrant 5 can be used Water content affects many characteristics of a substance or as titrating reagent. If the two-component reagent is used, Hydranal product; shelf life, production and application properties are some Methanol is replaced with Hydranal Solvent in the titration vessel. of them. Cosmetic products are often preparations with very high Product Application Method water content. For determining water content with Karl Fischer (KF) Panthenol Methanol : Chloroform 1:1 titration, precise sampling, weighing and administering of the sample to the titration cell are required. Handling the sample with syringe Titration at 50 °C and needle and differential weighing is the easiest and most precise Azulene mask 30 mL Methanol way and is therefore preferred. Tanning milk 30 mL Methanol For such samples with high water content, the volumetric KF method Suntan lotion LSF 8 Methanol : Chloroform 1:1 is recommended. The coulometric method is too sensitive for most Titration at 50 °C of these products. Products consisting of oil-water emulsions require Face tonic 30 mL Methanol an addition of chloroform; waxy substances can be dissolved more Foot bath 30 mL Methanol rapidly by titrating at elevated temperatures. Also, an addition of Glycerin 30 mL Methanol formamide can help dissolve some products and prevent a coating Liquid lanolin Methanol : Chloroform 1:1 of the indicator electrode. Titration at 50 °C Water-based cosmetic preparations such as facial tonics, suntan oils, Table 1 Application Overview of Selected Cosmetic Products shower gels and herbal shampoos, as well as beeswax preparations, (Excerpt from Application Report L 172) are analyzed in methanolic media by following the standard titration procedures (Application Report L 172). Emulsions such For all applications, Hydranal Methanol Dry or Hydranal Methanol as suntan creams or night creams should be analyzed at elevated Rapid can be used. temperatures in the presence of chloroform. The same applies to panthenol and liquid lanolin. Waxes that contain citric acid distearyl Recommended Procedure ester or pentaerythrite-di-coconut ester dissolve very slowly at 30 mL Hydranal Methanol dry or Hydranal Methanol Rapid (or the room temperature. We were able to precisely measure their water mixture recommended in Table 1) is filled into the titration vessel content with KF titration at 50 °C in the presence of trichloroethylene and titrated to dryness with Hydranal Composite 5. The required (Application Reports L 132, L 133). sample size is then added and the water content titrated with Hydranal Composite 5. Volumetric KF Applications for Cosmetic Products Hydranal LipoSolver CM is an ideal replacement for the methanol- Cosmetics usually require the addition of extra solvents due to their chloroform 1:1 mixture. components. We have analyzed a number of cosmetic products and

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22 Analytix | 5: 2015

Hydranal Application Report L 492

Sigma-Aldrich Laborchemikalien GmbH, Technical Service HYDRANAL Wunstorfer Str. 40, 30926 Seelze / Germany Tel.: +49 51 37 82 38 353 Fax: +49 51 37 82 38 698 [email protected]

L 492

Determining the Water Content in Hand Cream (Hand Balm)

The properties of this sample did not permit a determination of the water content under standard working conditions. The sample was not sufficiently distributed, it was impossible to detect the sample water completely. Parts of the sample coated the indicator electrode, and interfered with the endpoint indication.

Various tests showed that heating the medium in the titration vessel improved the distribution of the sample. The addition of formamide as a solubilizing agent additionally prevented the sample from coating the indicator electrode, and enabled a precise determination.

For determination of this sample, it is also possible to replace the toxic methanol and fill the titration vessel with HYDRANAL CompoSolver E. However, in this ethanol-based medium, only one portion of sample can be titrated in one filling of working medium.

Volumetric procedure

Fill the titration vessel with 30 mL HYDRANAL Methanol Dry, HYDRANAL Methanol Rapid or HYDRANAL CompoSolver E plus 10 mL HYDRANAL Formamide Dry. Heat the vessel (double- walled glass cell connected to a thermostat) to 50 °C and titrate to dryness with HYDRANAL Composite 5 (conditioning mode).

Weigh in a sample of 40 mg exactly (by differential weighing), and titrate the water content with HYDRANAL Composite 5.

Note

The two-component reagents HYDRANAL Titrant and HYDRANAL Solvent can also be used (with the same procedure as described above).

For titer determination, we recommend HYDRANAL Water Standard 10.0. It is liquid and therefore easy to handle with syringe and needle.

Reagents

34806 HYDRANAL Composite 5 37817 HYDRANAL Methanol Rapid 34741 HYDRANAL Methanol Dry 34734 HYDRANAL CompoSolver E 34724 HYDRANAL Formamide Dry

34801 HYDRANAL Titrant 5 34800 HYDRANAL Solvent

34849 HYDRANAL Water Standard 10.0

Seelze, 30.03.2004

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FeatureTitration Article 23

Water Content Recommended Sample Size Cat. no. Description 0.1% 3 –5 g Reagents for Volumetric One-Component Titration 0.5% 2 g 34805 Hydranal Composite 5 10% 250 mg 34806 Hydranal Composite 2 30% 100 mg 34734 Hydranal CompoSolver E 50% 80 mg 37817 Hydranal Methanol Rapid 80% 50 mg 34741 Hydranal Methanol Dry Table 2 Recommended Sample Sizes (in Relation to Sample Water Content, Reagents for Volumetric Two-Component Titration Calculated for Titer 5) 34801 Hydranal Titrant 5 34811 Hydranal Titrant 2 Application Reports (PDF) 34800 Hydranal Solvent Toothpaste (L 29) 34732 Hydranal Titrant 5E Wax for Cosmetic Use (L 132, L133) 34723 Hydranal Titrant 2E Cosmetics (L 172) 34730 Hydranal Solvent E Shampoo/Shower Gel (L 175) Auxiliaries and Reagents Containing Solubilizing Agents Calcium Glycerophosphate (L 251) 37855 Hydranal LipoSolver CM Gel-Emulsions (L 411) 34812 Hydranal Solvent CM Roll-on Deodorant (L 453) 34724 Hydranal Formamide Dry Hand Balm/Cream (L 492) 37863 Hydranal Chloroform Table 3 Available Application Reports for Cosmetic Products Table 4 Reagents for Volumetric KF Titration (selection)

More detailed information can be obtained from the respective Application Reports (designated L xxx). Sigma-Aldrich® offers over 700 application reports for KF titration. A complete list can be found on our website sigma-aldrich.com/hydranal. Application reports are available only on request; please contact us at [email protected] or use the contact form on our website. Technical Support Take advantage of our expertise, gained from over 35 years of experience, and our extensive applications database on KF titration. We’ll be glad to provide you with support for the analysis of your samples. Contact our Hydranal specialists (details below) or visit us at sigma-aldrich.com/hydranal

Questions regarding KF titration? Contact us at [email protected]

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