Analytix 4/2014, Proficiency Testing

Analytix

Issue 4 • 2014

Proficiency Testing – A Critical Tool

• Proficiency Testing Portal • NEW Flavor & Fragrance Standards • Analytical Standards of Boswellic Acids • Environmental Monitoring of Pharmaceuticals • Clinical Testing of Hormone Levels • Solvents for MS Applications • Carbonate-selective Sensors 2

Proficiency Testing – A Critical Tool

Dear Colleagues, Every year billions of measurements are performed at thousands of testing laboratories worldwide. These tests may include stability testing to

Editorial ensure the shelf-life of a pharmaceutical product, microbiological testing to ensure the safety of food products or environmental testing to monitor the quality of drinking water, to mention but a few.

Laboratories have to critically assess their ability to perform these measurements. Inaccurate measurements could lead to potential legal action and will certainly lead to loss of accredited status, customers and revenue.

One of the most useful tools available to assess the laboratory’s ability to perform these measurements is regular participation in a profi ciency testing program. Profi ciency testing provides an opportunity for laboratories to receive an independent appraisal of their data compared Patrick Brumfield Product Manager Proficiency Testing against a reference value and the performance of peer laboratories. [email protected] In an eff ort to deliver the best tools to our laboratory customer, we have developed a new testing portal which can be accessed at sigma-aldrich.com/proficiencytesting. This portal was designed with speed and simplicity as the focus, while providing the tools and features that PT customers need and have come to expect from their providers. You can read more about the specifics of this new portal further down in this issue.

To fi nd more information about our profi ciency testing program and to see the products that Sigma-Aldrich off ers for profi ciency testing please visit our webpage located at sigma-aldrich.com/profi ciencytesting

Sigma-Aldrich RTC has been operating proficiency testing programs since 1994 and is an accredited ISO 17043 PT Provider.

Best regards,

Patrick Brumfi eld Product Manager Profi ciency Testing

Analytix is published fi ve times per year by Sigma-Aldrich Chemie GmbH, MarCom Europe, Industriestrasse 25, CH-9471 Buchs SG, Switzerland Publisher: Sigma-Aldrich Marketing Communications Europe Publication Manager: Michael Jeitziner Editor: Daniel Vogler

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Feature Article Chromatography

4 New Data Entry Portal for Proficiency Testing 14 Choosing the Right Solvent for your Application …and Avoiding Signal Suppression in MS Standards Table of Contents Table 5 Flavor & Fragrance Standards Sensorics More than 100 NEW Flavor & Fragrance Standards for the Food and Cosmetic Industry 17 A Remarkable Carbonate-Selective Ionophore for the Determination of 6 Getting to the Heart of Frankincense Oceanic Carbon Dioxide Using an Ion- Analytical Standards of Boswellic Acids and Their Selective Electrode Separation

8 Pharmaceutical Environment Certified Reference Materials for Environmental Spectrometry Analysis of Pharmaceuticals 18 A New Approach for the Detection of Acidic 10 Short Overview of Sigma-Aldrich’s Isotope Pesticides in Water by MS Labeled Pesticide Standards Increasing the Sensitivity in Pesticide Analysis by Internal Standards for Food and Environmental Paired-Ion ESI Detection Analysis

11 Organic TraceCERT® CRMs Titration The Newest Product Additions to our Organic Neat Certified Reference Material Portfolio 21 Water Determination in Pharmaceutical Compounds 12 New & Emerging Clinical Testing Karl Fischer Titration with HYDRANAL® Reagents – Applications: Monitoring Hormone Levels Request the new Hydranal Product Line Overview during Treatment of Pain Brochure Solution CRMs for Corticosteroids, Progestogens and Other Hormones

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New Data Entry Portal for Proficiency Testing

Pat Brumfield, Product Manager Proficiency Testing [email protected] Feature Article Feature

Sigma-Aldrich® introduces our new proficiency-testing Previous Study Data/Reports data-entry portal. The new portal can be accessed at All of your previous study data will be transferred over to the sigma-aldrich.com/proficiencytesting and will be new system. This will allow for historical trending of results – available in mid-July 2014. This new portal will replace the a feature that will be available soon on the new portal. data-entry portal located at www.rt-corp.com Webinars on the new Portal The new portal has been streamlined and will off er superior A webinar will be off ered to demo the new portal and fi eld speed and ease of use. any questions that you might have. If you don’t have the opportunity to attend the webinar, a copy will be posted Sigma-Aldrich committed resources starting in late 2013 to on the homepage to view at your convenience. design a user interface that was effi cient, simple and some- thing that would meet the evolving needs of the end user. Please contact Pat Brumfi eld (pat.brumfi [email protected]) to The result is our new data-entry portal which is easy to sign up for our webinar taking place on Monday, October 20, learn, easy to remember and easy to use. 2PM Greenwich Mean Time.

Some of the features of the new Web portal: • One-click design that is easy to navigate, even for first time users • All open studies prominently displayed on the home screen • Easily add new personnel/analysts to your lab • Add accreditors and enter data all on a single page • Only the applicable methods are displayed in the method drop-down box • Easily copy down method/analyst/analysis date to all analytes in a sample • Data is actively saved as you report • A confirmation screen that shows exactly what you have entered and will be submitted for evaluation • The ability to import LIMS files rather than manually entering your results

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Flavor & Fragrance Standards More than 100 NEW Flavor & Fragrance Standards for the Food and Cosmetic Industry

Eva Katharina Richter, Product Manager Analytical Standards [email protected]

Sigma-Aldrich® provides a broad range of flavor and fra- pounds and flavor enhancers as well as EU banned sub- grance standards for quality control of food and cosmetic stances. Test your products against our Flavor & Fragrance products. Our large portfolio focuses on analytical standards Standards to ensure they maintain their high quality! Find an for aroma compounds from the “EU positive list of autho- up-to-date product list of all Flavor & Fragrance Standards rized fl avoring substances” (EFSA 10/2012) and the “EU-Reg- arranged according to their occurrence in food and bever- ulation on cosmetic products” (1223/2009). Our selection, ages, by substance classifi cation, and in alphabetical order which is continuously growing, includes allergenic com- at sigma-aldrich.com/fl avor

Cat. No. Brand Description Package Size Cat. No. Brand Description Package Size 67369 Fluka® (−)-Ambroxide 100 mg 49919 Fluka Dimethyl anthranilate 1 mL 91034 Fluka (−)-Caryophyllene oxide 100 mg 68986 Fluka Dimethyl disulfide 1 mL 54328 Fluka (−)-Globulol 10 mg 04011 Fluka Dimethyl malonate 1 mL 43201 Fluka (−)-Isoledene 100 mg 95563 Fluka Ethyl 2-trans-4−decadienoate 25 mg 93483 Fluka (+)-Cedrol 100 mg 63954 Fluka Ethyl acetoacetate 1 mL 44889 Fluka (+)-Nootkatone 50 mg 72505 Fluka Ethyl benzoate 1 mL, 5 mL 18032 Fluka (1R)-(–)-Myrtenal 1 mL 66761 Fluka Ethyl cinnamate 100 mg

56699 Fluka (3aR)-(+)-Sclareolide 100 mg 08996 Fluka Ethyl lactate 1 mL Standards 04969 Fluka (S)-(−)-Perillaldehyde 1 mL 73539 Fluka Ethyl linoleate 1 mL 04174 Fluka 1-Acetonaphthone 1 mL, 5 mL 91224 Fluka Ethyl phenylacetate 1 mL, 5 mL 75544 Fluka 1-Dodecanol 1 mL, 5 mL 43348 Fluka Farnesol 1 mL 18698 Fluka 1-Methylpyrrole 1 mL, 5 mL 89985 Fluka gamma-Undecalactone 1 mL 76723 Fluka 1-Penten-3-one 1 mL 40951 Fluka gamma-Valerolactone 1 mL 50714 Fluka 2,3,5-Trimethylpyrazine 1 mL 61696 Fluka Heptaldehyde 1 mL 43042 Fluka 2,3-Benzofuran 1 mL 59964 Fluka Histamine 100 mg 42038 Fluka 2,3-Butanediol 250 mg 80725 Fluka Hydrocinnamic acid 100 mg 56931 Fluka 2,6-Diisopropylphenol 1 mL 66010 Fluka Hydroxycitronellal 1 mL 18478 Fluka 2-Acetonaphthone 100 mg 95668 Fluka Isoamyl octanoate 1 mL, 5 mL 55752 Fluka 2-Ethoxynaphthalene 100 mg 94888 Fluka Isobutyl butyrate 1 mL, 5 mL 69530 Fluka 2-Ethylfuran 1 mL, 5 mL 06755 Fluka Isobutylamine 1 mL 14249 Fluka 2-Furyl methyl ketone 1 mL 07055 Fluka Isoeugenyl acetate 100 mg 18245 Fluka 2'-Hydroxyacetophenone 1 mL 68853 Fluka Isoquinoline 1 mL 94414 Fluka 2-Methoxybenzaldehyde 100 mg 49599 Fluka Linalyl acetate 100 mg 61904 Fluka 2-Methylbutyraldehyde 100 mg 07406 Fluka m-Anisaldehyde 1 mL 92015 Fluka 2-Naphthalenethiol 100 mg 95401 Fluka Menthone, mixture of isomers 1 mL, 5 mL 14672 Fluka 2-Pentyl butyrate 1 mL 41263 Fluka Methyl benzoylformate 1 mL 74350 Fluka 2-Picoline 500 mg 80835 Fluka Methyl cinnamate 250 mg 44638 Fluka 3-(Methylthio)propionaldehyde 500 mg 52466 Fluka Octanal 1 mL, 5 mL 92588 Fluka 3,4-Dihydroxybenzaldehyde 100 mg 08066 Fluka Oleyl alcohol 100 mg 94415 Fluka 3-Carene 1 mL, 5 mL 92819 Fluka o-Tolualdehyde 1 mL 43212 Fluka 3-Heptanol 1 mL 94666 Fluka p-Anisic acid 100 mg 04649 Fluka 3-Hexanol 1 mL 66962 Fluka Pentyl acetate 1 mL 93856 Fluka 3-Octanol 1 mL 73747 Fluka Phenethyl acetate 1 mL, 5 mL 05339 Fluka 3-Picoline 1 mL 30311 Fluka Phenyl acetate 1 mL 43045 Fluka 3-Pyridinemethanol 1 mL 92528 Fluka Phenyl disulfide 100 mg 90034 Fluka 4-Hydroxy-3-methoxycinnamic acid 100 mg 91897 Fluka Phloretin 10 mg 43919 Fluka 4-Methylthiazole 1 mL, 5 mL 61074 Fluka Propiophenone 1 mL, 5 mL 67397 Fluka 6-Methyl-5-hepten-2-one 1 mL 53646 Fluka Propylamine 1 mL 07056 Fluka Allyl cinnamate 1 mL 41423 Fluka p-Tolualdehyde 1 mL 12448 Fluka alpha-Humulene 250 mg 49817 Fluka Pyrrole 1 mL, 5 mL 30627 Fluka alpha-Terpineol 100 mg 94517 Fluka Quinoline 1 mL 68518 Fluka Amylamine 1 mL 42993 Fluka Theobromine 100 mg 95054 Fluka Azelaic acid 100 mg 92358 Fluka Thioanisole 1 mL 80024 Fluka Benzoin 100 mg 03416 Fluka Thymoquinone 100 mg 89983 Fluka Benzyl isothiocyanate 500 mg 05549 Fluka trans-2,cis-6-Nonadienal 100 mg 08900 Fluka Benzyl mercaptan 1 mL 90244 Fluka trans-2-Heptenal 100 mg 78466 Fluka Benzyl propionate 1 mL, 5 mL 07592 Fluka trans-2-Nonenal 1 mL 75031 Fluka Butyl anthranilate 1 mL 52464 Fluka trans-2-Octenal 1 mL, 5 mL 68119 Fluka Butyl propionate 1 mL, 5 mL 94575 Fluka trans-2-Pentenal 1 mL 90893 Fluka Butylamine 1 mL, 5 mL 74103 Fluka Triacetin 1 mL, 5 mL 94194 Fluka Butyraldehyde 1 mL 43103 Fluka Triethyl orthoformate 1 mL, 5 mL 52063 Fluka Cadaverine 1 mL 73487 Fluka Triethylamine 1 mL, 5 mL 43646 Fluka Citronellyl acetate 1 mL 80139 Fluka Undecanal 1 mL 49673 Fluka Dibenzyl ether 1 mL, 5 mL 94490 Fluka δ-Octalactone, mixture of isomers 1 mL, 5 mL 91969 Fluka Diethyl carbonate 1 mL, 5 mL Table 1 NEW Flavor & Fragrance Standards

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Getting to the Heart of Frankincense Analytical Standards of Boswellic Acids and Their Separation

Matthias Nold, Product Manager Analytical Standards [email protected] Hugh Cramer, Application Scientist [email protected]

The trees of the genus boswellia have been used in tradi- are a group of pentacyclic triterpene acids of which tional medicine for thousands of years as an anti-infl amma- E-boswellic acids are the major biologically active constitu- tory agent. Its resin is known as frankincense or olibanum ents. Sigma Aldrich® off ers six boswellic acids as analytical and is still widely used in dietary supplements and in herbal standards and two neat standards of D+E mixtures for use medicinal products. The most characteristic components in research and quality control.

H3C CH3 CH3 CH3 H3C CH H C CH 3 3 3 CH H C 3 H C 3 CH3 H 3 CH3 H H C 3 CH3 H O H CH H CH3 3 HO H CH3 HO H3C O Standards H C H H3C H 3 H3C H O OH O OH O OH 80342 06813 49873 E-boswellic acid D-boswellic acid 3-O-Acetyl-E-boswellic acid

CH3 CH3 H3C CH3 H C H3C 3

CH3 CH3 CH3 O O H C H C H C 3 CH3 H 3 CH3 H 3 CH3 H O O H CH H CH3 H CH 3 H C O 3 H3C O 3 HO H C H H3C H 3 H3C H O OH O OH O OH

56208 74607 78535 3-O-Acetyl-D-boswellic acid 3-O-Acetyl-11-keto-E-boswellic acid 11-keto-E-boswellic acid

Figure 1 Chemical Structures of the Boswellic Acids

Separation of Boswellic Acids Several conditions, using a variety of solid phases, have been Figure 2 shows the chromatograms of the ethanolic screened for their suitability to separate six boswellic acids. extract of a boswellia herbal supplement as well as of a The shortest analysis time was achieved using an Ascentis mixture of the six analytical standards. Express Column (2.7 μm particle size, L × I.D. 10 cm × 3.0 mm) and acetonitrile/phosphoric acid as eluents.

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Conditions

Column: Ascentis Express Phenyl-Hexyl, 10 cm x 3.0 mm I.D., 2.7 μm (53345-U) Mobile Phase: (A) 0.1% phosphoric acid; (B) acetonitrile Gradient: 50% B for 0.5 min; to 100% B in 4.5 min; held at 100% B for 5 min Flow Rate: 0.6 mL/min Pressure: 2390 psi (165 bar) Column temp.: 35 °C Detector: UV at 210 nm Injection: 5 μL Sample(s): 6 Boswellic acids 50μg/mL in 70:30, water:methanol extract in 50:50, water:ethanol Extraction: 1. Weigh 100 mg Bosewellia Herbal Supplement 2. Add 10 mL ethanol 3. Sonicate for 30 min at 40 °C A Boswellia Extract 4. Dilute: 100 μL extract in ethanol, 400 μL ethanol, 500 μL water

Peak IDs

1) 11-Keto-β-boswellic-acid 2) 3-O-Acetyl-11-keto-β-boswellic-acid 3) α-boswellic-acid Standards 4) β-boswellic-acid 5) 3-O-Acetyl-α-boswellic-acid 6) 3-O-Acetyl-β-boswellic-acid

B Analytical Standards at 50Pg/mL of Each Component

Figure 2 Experimental Conditions and Chromatograms of the Separation of Boswellic Acids

Cat. No. Brand Description Package Size 74607 Fluka® 3-O-Acetyl-11-keto-β-boswellic acid 5 mg 56208 Fluka 3-O-Acetyl-α-boswellic acid 5 mg 49873 Fluka 3-O-Acetyl-β-boswellic acid 5 mg 96729 Fluka 3-O-Acetylboswellic acid, mixture of α and β 5 mg 06813 Fluka α-Boswellic acid 10 mg 80342 Fluka β-Boswellic acid 5 mg 63850 Fluka Boswellic acid, mixture of α and β 5 mg 78535 Fluka 11-Keto-β-boswellic acid 5 mg 53345-U Supelco® Ascentis Express Phenyl-Hexyl, 2.7 Micron HPLC Column 10 cm × 3.0 mm

Table 1 Analytical Standards of Boswellic Acids and Column Used

Sigma-Aldrich continuously expands its off ering of analytical On our Web page at sigma-aldrich.com/medicinalplants, standards for plant constituents, intended for the analysis of you will fi nd an up-to date list of all phytopharma standards medicinal plants and herbal medicinal products. sorted in alphabetical order, by substance class and by genus of a large number of the most popular medicinal plants.

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Towards a Healthier Environment Certified Reference Materials for Environmental Analysis of Pharmaceuticals

Matthias Nold, Product Manager Analytical Standards [email protected]

Therefore, the occurrence and impact of pharmaceutical substances in the environment is gaining increasing attention from researchers and authorities, as well as from com- panies in the pharmaceutical industry, which have an inter- est in minimizing their ecological footprint. This increasing attention was also reflected by the huge quantity of ses- sions and posters on the topic at this year’s SETAC annual meeting[1]. Many studies support the presumption that active pharmaceutical substances (API) do have a detrimen- tal eff ect on the ecosystem and that their release into the environment needs to be monitored and controlled.

Authorities such as the EPA (U.S. Environmental Protection Without a doubt, we can attribute our present-day quality Agency)[2] or the German Umweltbundesamt (UBA)[3] per- Standards of life and long life expectancy largely to the achievements form and/or support studies in this field with the aim of of the medical profession during the last century. Nowa- establishing appropriate guidelines. The Water Framework days, huge quantities of pharmaceutical substances are Directive[4] of the EU also has an eye on pharmaceuticals, and produced and consumed. However, either through excre- in 2012, added diclofenac, which was the fi rst pharmaceutical tion or improper disposal via the toilet, a lot of pharmaceu- to be added to the list of proposed priority substances. ticals end up in sewage treatment plants and eventually in ground and surface waters. This environmental presence of highly active chemicals may have unforeseeable eff ects on the ecosystem.

Cat. No. Brand Description Package Size PHR1005 Fluka® Acetaminophen 1 g PHR1127 Fluka Amoxicyllin trihydrate 1 g PHR1067 Fluka Carbamazepine 1 g PHR1412 Fluka Chloramphenicol 1 g PHR1167 Fluka Ciprofloxacin 1 g PHR1038 Fluka Clarithromycin 500 mg PHR1058 Fluka Clotrimazole 1 g PHR1144 Fluka Diclofenac sodium salt 1 g PHR1145 Fluka Doxycycline hyclate 1 g PHR1039 Fluka Erythromycin 1 g PHR1246 Fluka Fenofibrate 500 mg PHR1049 Fluka Gabapentin 1 g PHR1004 Fluka Ibuprofen 1 g PHR1247 Fluka Indomethacin 500 mg PHR1084 Fluka Metformin hydrochloride 500 mg PHR1076 Fluka Metoprolol tartrate 1 g PHR1040 Fluka Naproxen 500 mg PHR1308 Fluka Propranolol hydrochloride 1 g PHR1389 Fluka Propyphenazone 1 g PHR1126 Fluka Sulfamethoxazole 1 g PHR1041 Fluka Tetracycline hydrochloride 500 mg PHR1056 Fluka Trimethoprim 1 g

Table 1 Some Pharmaceuticals with Potential Relevance for Environmental Analysis and Proposed CRMs

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Several aspects need to be considered for the priorisation To complement this offering, Sigma-Aldrich also offers a of pharmaceuticals for environmental monitoring: The comprehensive portfolio of pharmaceutical metabolites ecotoxicological potential, the occurence in the aquatic and isotope labeled pharmaceuticals (sigma-aldrich.com/ environment and the annual consumption of the drug. pharmametabolites) as presented in an article in the previous issue of Analytix[5]. To ensure reliable and traceable quantitative results, ISO 17025 accredited labs should use proper certifi ed reference materi- References als (CRMs). For this purpose, Sigma-Aldrich® off ers a compre- [1] www.basel.setac.eu/?contentid=763 [2] www.epa.gov/ppcp/ hensive portfolio of pharmaceutical compounds (APIs, related [3] Bergmann, A. et al. Zusammenstellung von Monitoringdaten substances and excipients) of pharmaceutical secondary zu Umweltkonzentrationen von Arzneimitteln UBA-FB 001525; standards. Though these products have been developed for 2011. use in pharmaceutical quality control and are traceable to [4] http://ec.europa.eu/environment/water/water-dangersub/ pharmacopoeia compendial standards, they are also certifi ed lib_pri_substances.htm#prop_2011_docs [5] Richter, E. Analytix 3/2014, page 8 reference materials by the defi nition of ISO, as they are manufactured under ISO/IEC17025 and ISO Guide 34 double accreditation, and a certifi ed value, including uncertainty, is given in addition to the pharmacopoeia traceable values. The list on the previous page shows some common pharmaceuticals and proposed certifi ed reference materials. The complete portfolio comprises over 400 products and can be found at

sigma-aldrich.com/pharmastandards Standards

Inorganic Custom Standards An Interactive Online Platform

With a few simple mouse clicks, you can defi ne For all TraceCERT Custom Standards we guarantee: your own multi-component standards for ICP and IC • Certification under double accreditation following in TraceCERT® quality, using our Inorganic Custom ISO/IEC 17025 and ISO Guide 34 Standards Online Platform: • Highest level of accuracy and reliability • Minimized uncertainties and lot-specific values sigma-aldrich.com/csp • Traceability to at least two independent references (i.e., NIST, BAM or SI unit kg) • Printed certificate according to ISO Guide 31 • Light and gas-tight aluminum foil bag packaging allowing up to four years’ shelf life

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Short Overview of Sigma-Aldrich’s Isotope Labeled Pesticide Standards Internal Standards for Food and Environmental Analysis

Eva Katharina Richter, Product Manager Analytical Standards [email protected]

Cat. No. Brand Description Package Size

39246 Fluka® Acetamiprid-d3 50 mg

34086 Fluka Alachlor-d13 10 mg 32440 Fluka Amitraz Metabolite BTS 27271 10 mg

(N/methyl-d3)

34053 Fluka Atrazine-d5 10 mg

32965 Fluka Bentazon-d7 10 mg

34164 Fluka Bis(2-ethylhexyl)phthalate-3,4,5,6-d4 25 mg

32413 Fluka Carbendazim-d3 10 mg

34019 Fluka Carbofuran-d3 10 mg

32232 Fluka 6-Chloro-2,4-diamino-1,3,5-triazine-13C3 10 mg

56816 Fluka Clothianidin-d3 50 mg 34021 Fluka 4,4’-DDT-d 10 mg The Sigma-Aldrich® pesticide standards product line is the most compre- 8 34169 Fluka Dibutyl phthalate-3,4,5,6-d 25 mg hensive portfolio available on the market. We proudly off er more than 4 NEW 32381 Fluka DEET-(diethyl-d10) 25 mg

Standards 1300 high-purity pesticide and pesticide metabolite standards and certi- 48049 Supelco 1,4-Dichlorobenzene-d4 solution; 1 mL fi ed reference materials for food and environmental analysis. 2000 μg/mL in methanol 442247 Supelco 1,4-Dichlorobenzene-d 1000 mg Our comprehensive portfolio includes several isotope-labeled pesticide 4 442228 Supelco 1,2-Dichloroethane-d 1000 mg standards. To determine matrix eff ects and result bias due to a loss of the 4 34233 Fluka Dicamba-d 10 mg analyte during the workup process, isotope-dilution mass spectrometry 3 34186 Fluka Dicyclohexyl phthalate-d4 10 mg, 25 mg is often applied. This technique takes advantage of the fact that isotope- NEW 13 32311 Fluka Diflubenzuron-(chlorophenyl- C6) 10 mg labeled compounds have nearly the same physical properties as the 32274 Fluka NEW 4-(3,6-Dimethyl-3-heptyl)phenol- 1 mL

non-labeled analogues, and thus the same behavior in the workup and 3,5-d2 solution; 100 μg/mL in acetone

sample-preparation process. However, they can be distinguished in mass 34201 Fluka Dimethyl-d6 phthalate 25 mg spectrometry. Therefore, by spiking the sample before workup with an 34018 Fluka Diuron-d6 10 mg 34214 Fluka DNC-d 10 mg isotope-labeled analogue, material loss during workup can be deter- 8 03811 Fluka α-HCH-d 5 mg mined and compensated. 6 34170 Fluka Imidacloprid-d4 10 mg

Please fi nd below a list of our current products. For more information, 34017 Fluka Isoproturon-d6 10 mg NEW please visit our Web page at sigma-aldrich.com/pesticides or order 32300 Fluka Methiocarb-(N-methyl-d3) 10 mg 32668 Fluka Nabam-d 10 mg our brand NEW brochure at sigma-aldrich.com/lit-request 4 48715-U Supelco Naphthalene-d8 solution; 1 mL 2000 μg/mL in methylene chloride 33452 Fluka Parathion-ethyl-d 10 mg Pesticide Standards 10 For Food & Environmental Analysis 34209 Fluka Pirimicarb-d6 10 mg

34054 Fluka Simazine-d10 10 mg NEW 13 43293 Fluka Tebuconazole-(trimethyl- C3)5 mg

38176 Fluka Thiamethoxam-d3 25 mg

33853 Fluka Thionazin-d10 10 mg

• Neat Standards • Single and Multi-Component 34023 Fluka Trichloroanisole-d 50 mg Solutions 5 • Certified Reference Materials (CRMs) • Matrix CRMs • Isotopically Labeled Internal Standards • Pesticide Metabolite Standards • Proficiency Testing Materials (PT) Table 1 Neats and Solutions for Isotope-Labeled Pesticides

D D D N D Cl O D NO2 D CD D N Cl 3 N H D Cl N CD3 D Cl S O N Cl O D Cl D H N Cl

Figure 1 Molecular Structure of Deuterated α-HCH Figure 2 Molecular Structure of Deuterated Bentazon Figure 3 Molecular Structure of Deuterated Imidacloprid

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Organic TraceCERT® CRMs The Newest Product Additions to our Organic Neat Certified Reference Material Portfolio

Matthias Nold, Product Manager Analytical Standards [email protected]

For accredited testing labs, the availability of reliable and In the last issue of Analytix, we presented 25 new products, traceable certifi ed reference materials (CRMs) is crucial since and we are now able to present the latest additions to the the use of CRMs for calibration is demanded by ISO/IEC rapidly growing organic TraceCERT product line. You can 17025. For the certification of Sigma-Aldrich®’s organic fi nd a list of the complete portfolio of over 150 products at TraceCERT products, high-performance quantitative NMR sigma-aldrich.com/organiccrm (HP-qNMR®) is applied as a relative primary method to achieve traceability to NIST SRM. If you would like to learn Cat. No. Brand Product Package Size more about qNMR and Sigma-Aldrich’s in-house capabili- 63634 Fluka® Acetophenone 50 mg ties in this fi eld, please refer to the references cited below. 14181 Fluka Dimethyl isophthalate 100 mg 97627 Fluka Monoethanolamine 100 mg The organic TraceCERT reference materials are character- 80026 Fluka 2,4,6-Tribromophenol 100 mg 67180 Fluka 1,2,4-Trichlorobenzene 100 mg ized by: 53766 Fluka 2,4,6-Trimethylphenol 100 mg • Certified content by quantitative NMR (qNMR) 30304 Fluka Vanillin 50 mg

• Manufactured under ISO/IEC 17025 / ISO Guide 34 50409 Fluka Thymol 1 g Standards double accreditation References • Superior level of accuracy, calculated uncertainties, and [1] Weber, M.; Hellriegel, C.; Rueck, A.; Sauermoser, R.; Wuethrich, J. lot-specific values Using high-performance quantitative NMR (HP-qNMR®) for certifying traceable and highly accurate purity values of • Traceability to NIST SRM organic reference materials with uncertainties <0.1 %, • Comprehensive documentation delivered with the Accred. Qual. Assur. 18. 2013, 91– 98 product (certification according to ISO Guide 31) [2] TraceCERT® Organic Certified Reference Materials, Analytix, Vol. 3 2010; Vol. 1 2011; Vol. 3 2011; Vol. 4 2011. [3] TraceCERT® Organic Certified Reference Materials, Analytix, Vol. 2 2010.

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

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

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New and Emerging Clinical Testing Applications: Monitoring Hormone Levels during Treatment of Pain Solution CRMs for Corticosteroids, Progestogens and Other Hormones

Derrell Johnson, Manager, New Product Strategy & Tactical Marketing [email protected] Maximilian Magana, Technical Marketing Specialist [email protected]

New clinical applications are emerging that monitor hormone levels for Cerilliant Hormone Certified Spiking Solutions® the diagnosis and treatment of pain. Pain management clinics, for Cat. No. Description Package Size example, are increasingly quantifying neurosteroids, corticosteroids, Corticosteroids and other hormones by LC-MS/MS and other analytical methods for A-096 Aldosterone 100 μg/mL in Acetonitrile indication of abnormal hormone production and its relationship to C-106 Cortisol 1.0 mg/mL in Methanol C-113 Cor tisol-D 100 μg/mL in Methanol severe pain.[1,2] According to one study, serum levels of hormones such 4 C-117 Corticosterone 1.0 mg/mL in Methanol as cortisol, pregnenolone, testosterone, thyroid, and dehydroepiandros- C-130 Cortisone 100 μg/mL in Methanol terone (DHEA) represent the most objective biomarkers of severe, D-061 11-Deoxycortisol 1.0 mg/mL in Methanol uncontrolled pain.[1] D-062 21-Deoxycortisol 100 μg/mL in Methanol

D-076 21-Deoxycortisol-D8 100 μg/mL in Methanol The study’s author stated that patients taking opioids for treatment of D-078 11-Deoxycortisol-D5 100 μg/mL in Methanol pain should be screened for hormone abnormalities. Several hormones, D-085 Dexamethasone 1.0 mg/mL in Methanol including cortisol, testosterone, and pregnenolone, are suppressed by D-105 11-Deoxycorticosterone 100 μg/mL in Methanol opioids with longer-acting opioids the most eff ective at hormone sup- Progestogens Standards pression. Replacement hormone therapy may be needed to counteract H-085 17α-Hydroxyprogesterone 1.0 mg/mL in Methanol the eff ect of opioids on serum hormone levels. For pain patients with H-096 17α-Hydroxyprogesterone-D8 100 μg/mL in Methanol H-100 17α-Hydroxyprogesterone-2,3,4-13C 100 μg/mL in Methanol abnormally low hormone levels that are not taking opioids, treatment 3 H-105 17α-Hydroxypregnenolone 100 μg/mL in Methanol with neuroregenerative hormones such as hCG or human chorionic P-069 Progesterone 1.0 mg/mL in Acetonitrile gonadotropin may provide a benefi t of pain reduction.[1] P-070 Progesterone-D9 100 μg/mL in Acetonitrile P-104 Pregnenolone Coming Soon [3] Testosterone and cortisol have also been implicated in neonatal and Dehydroepiandrosterone (DHEA) [4,5] adult pain-related stressors. In these studies, testosterone and cortisol D-063 DHEA 1.0 mg/mL in Methanol

levels in blood, saliva, or hair were determined using immunoassay and D-064 DHEA-D5 100 μg/mL in Methanol related analytical techniques. The fi ndings from these studies demon- D-065 DHEAS 1.0 mg/mL (as free sulfate) in strate the importance and infl uence of cortisol and testosterone levels Methanol D-066 DHEAS-D 100 μg/mL (as free sulfate) in on pain and pain thresholds during times of high stress.[3,4,5] 5 Methanol Testosterone To view Cerilliant’s extensive catalog of solution Certified Reference D-073 5α-Dihydrotestosterone (DHT) 1.0 mg/mL in Methanol Materials (CRMs), including hormone categories such as corticosteroids, D-077 5α-Dihydrotestosterone-D3 100 μg/mL in Methanol DHEA, progestogens, testosterone, and thyroid, visit H-059 6β-Hydroxytestosterone 100 μg/mL in Methanol

sigma-aldrich.com/cerilliant T-034 6β-Hydroxytestosterone-D3 100 μg/mL in Methanol T-037 Testosterone 1.0 mg/mL in Acetonitrile

Register at sigma-aldrich.com/registercerilliant to receive news on T-046 Testosterone-D3 100 μg/mL in Acetonitrile 13 the latest reference standards being developed by Cerilliant. T-070 Testosterone-2,3,4- C3 100 μg/mL in Acetonitrile Thyroid

T-073 L-Thyroxine (T4) 100 μg/mL in 0.1N NH3 in Methanol

T-074 3,3',5-Triiodo-L-thyronine (T3) 100 μg/mL in 0.1N NH3 in Methanol

T-075 3,3',5'-Triiodo-L-thyronine (Reverse T3) 100 μg/mL in 0.1N NH3 in Methanol 13 13 T-076 L-Thyroxine- C6 (T4- C6) 100 μg/mL in 0.1N NH3 in Methanol 13 13 T-077 3,3',5-Triiodo-L-thyronine- C6 (T3- C6) 100 μg/mL in 0.1N NH3 in Methanol 13 T-078 3,3',5'-Triiodo-L-thyronine- C6 100 μg/mL in 0.1N NH3 in Methanol 13 (Reverse T3- C6)

References [1] Hughes, D. Hormone Levels Can Validate Presence of Pain and Affect Treatment. Accessed 11 April 2014 from: www.empr.com/hormone-levels-can-validate-presence-of-pain-and-affect-treatment/article/310528/ [2] Tennant, F. The Use of Hormones in Chronic Pain. Accessed 11 April 2014 from: www.journalofprolotherapy.com/pdfs/issue_08/issue_08_06_hormones_for_pain.pdf [3] Grunau, R. E.; Cepeda, I. L.; Chau C. M. Y.; Brummelte S.; Weinberg J.; Lavoie P. M.; Ladd M.; Hirschfeld A. F.; Russell E.; Koren G.; Van Uum S.; Brant R.; and Turvey S. E. Neonatal Pain-Related Stress and NFKBIA Genotype Are Associated with Altered Cortisol Levels in Preterm Boys at School Age. Accessed 29 May 2014 from: www.ncbi.nlm.nih.gov/pmc/articles/PMC3774765/#__ffn_sectitle [4] Choi J. C.; Chung M. I.; and Lee Y. D. Modulation of pain sensation by stress-related testosterone and cortisol. Accessed 29 May 2014 from: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2044.2012.07267.x/pdf [5] Karlén J.; Ludvigsson J.; Frostell A.; Theodorsson E.; and Faresjö, T. Cortisol in hair measured in young adults – a biomarker of major life stressors? Accessed 29 May 2014 from: www.biomedcentral.com/1472-6890/11/12#B37

sigma-aldrich.com/cerilliant 13

New TLC Staining Solution for Boronic Acids

Curcumin solution 28982 eff ectively 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; Standards 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; 3 346225 (2-Methylpropyl)-boronic acid; For more information on TLC derivatization, visit sigma-aldrich.com/derivatization 4 683590 Potassium trans-3-methoxy-1- propenyltri-fluoroborate Reference: [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.

New! Brochure for Cosmetic Standards For Beauty with Safety

For precise quality control of raw materials or formulated personal-care and cosmetic products, we off er analytical standards for several groups of ingredients, including:

• Antibacterial, antifungal substances • PAHs • Colorants • Plasticizers • Emulsifiers, thickeners, opacifiers • Preservatives • Fragrances • Propellants • Metals • UV blockers

Order the brand NEW brochure or visit sigma-aldrich.com/cosmetics for an up-to-date product list.

sigma-aldrich.com/derivatization 14

Choosing the Right Solvent for Your Application …and Avoiding Signal Suppression in MS

Matthias Drexler, Product Manager, Analytical Reagents and Spectroscopy [email protected] Experimental data courtesy of Otto Savolainen and Alastair Ross at Chalmers University of Technology, Gothenburg, Sweden

Introduction Results In routine LC-MS analysis, reliability of methods is crucial in Figure 1 clearly shows all key analytes are separated, ionized order to avoid false analytical data or increase in time per and detected with the selected method when utilizing a sample due to necessary corrections or repetition of deter- gradient of mQ water and LC-MS CHROMASOLV methanol. minations. Besides instrument sensitivity, which generally improves with every generation, solvent quality plays an The instrument was then run with two blanks containing increasingly important role. Lesser-grade solvents may con- standard HPLC grade methanol, after which the same ana- tain impurities, and are not tested for LC-MS applications, lyte mixture was analyzed again. Immediately strong signal resulting in interferences or signal suppression. Impurities suppression can be seen in the resulting chromatogram that most commonly cause problems are high alkali-ion (see Figure 2). The eff ect is especially apparent for the ana- concentrations, plasticizers and detergents. lytes with longer retention time. After 10 injections working with HPLC grade methanol, the suppression effects are Experimental even higher (Figure 3) leading to complete suppression of A mixture containing 6 alkylresorcinols (phenolic lipids some of the analyte signals after a few dozen injections. used as food intake biomarkers) at 0.1 μg/mL was separated The instrument becomes “blind” to the analytes at these on a Titan C18 UHPLC column, using a previously published concentrations. method (Ross 2012). [1] After switching back to a gradient containing LC-MS grade The following conditions were employed, with determina- methanol, two blanks were run before injection of the analyte tions using fi rst LC-MS CHROMASOLV® methanol, then stan- mix and running the method again. In Figure 4, only minimal dard HPLC grade methanol, before switching back to LC-MS (if at all) remaining signal suppression can be seen and all

Chromatography methanol. The standard mixtures were created in neat 6 analytes can again be clearly detected and quantifi ed. methanol. Conclusion Column Supelco Titan™ C18 1.9 μm 2.1x100 mm These results show the importance of choosing the right Mobile phase A = mQ water, B = MeOH (LC-MS solvent quality to support the analytical task at hand. High CHROMASOLV or standard HPLC grade) purity and sensitive UV gradient methods can give only a Gradient min %B rough picture of a solvent and its suitability for MS detec- 089 tion. Relying on sub-par solvents, especially during method 699 development, may lower the apparent cost per sample, but 799 889have a serious negative impact on limit of quantification 10 89 (LOQ), while also negatively affecting the lifetime of the Flow 0.65 mL/min instrument. Injection volume 1 μL MS APCI+ operated in MRM mode Our LC-MS CHROMASOLV solvents are application tested, Instrument Shimadzu LCMS-8030plus including Nexera off er low cation impurities, and high reliability to help sci- LC-30AD pumps, Nexera SIL-30AC autosampler, entists to avoid the hidden costs resulting from signal sup- Nexera CTO-20AC column oven, Prominence DGU-20A 5r degasser and Shimadzu 8030plus pression. More information can be found at triple quadrupole sigma-aldrich.com/lc-ms

sigma-aldrich.com/lc-ms 15

(x1,000) 4,00

3,75

3,50

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3,00

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3,5 1,50

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1,5 0,50 1,0 0,25 0,5 0,00 0,0

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 min 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 min Figure 1 Chromatogram Using LC-MS CHROMASOLV Methanol in the Gradient Figure 2 Chromatogram after 2 Blanks Run and First Injection using Standard

(x1,000) HPLC Grade Methanol

3,50

(x1,000) 3,25 4,00

3,00 3,75

2,75 3,50

3,25 2,50 3,00 2,25 2,75

2,00 2,50

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1,00 1,25

0,75 1,00

0,75 0,50 0,50 0,25 0,25

0,00 0,00

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 min 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 min Figure 3 Chromatogram after 10 Injections using a Gradient Containing Standard Figure 4 Chromatogram after Switching back to a Gradient with LC-MS HPLC Grade Methanol CHROMASOLV Methanol and Running Two Blanks

For very sensitive UHPLC-MS instruments, we recommend using LC-MS To learn more about LC-MS Ultra, visit sigma-aldrich.com/uhplc

Ultra solvents, which are tested on UHPLC-(qTOF)MS both in positive Chromatography References: and negative mode, are fi lled in leach-resistant clear borosilicate bottles [1] Ross, AB. Analysis of alkylresorcinols in cereal grains and products using for very low cation concentrations, and exhibit an extremely strong lot- ultra high-pressure liquid chromatography with fluorescence, ultraviolet, to-lot consistency to get the most out of today’s sensitive UHPLC-MS and CoulArray electrochemical detection. Journal of Agricultural and Food instruments. Chemistry 60, 2012, 8954 –8962.

Cat. No. Featured and Related Products Package Size Solvents 34967 Acetonitrile, LC-MS CHROMASOLV 250 mL, 1 L, 2.5 L, 4x4 L, 6x1 L, 4x2.5 L, 20 L 34966 Methanol, LC-MS CHROMASOLV 1 L, 2.5 L, 4x4 L, 6x1 L, 4x2.5 L 39253 Water, LC-MS CHROMASOLV 1 L, 4x4 L, 20 L 14261 Acetonitrile, LC-MS Ultra CHROMASOLV, for UHPLC-MS 1 L, 2 L 14262 Methanol, LC-MS Ultra CHROMASOLV, for UHPLC-MS 1 L, 2 L 14263 Water, LC-MS Ultra CHROMASOLV, for UHPLC-MS 1 L, 2 L Columns and Accessories 577124-U Titan C18 UHPLC Column, 1.9 micron, 10 cm x 2.1 mm 1 ea 577127-U Titan C18 UHPLC Guard Cartridge, 1.9 micrometer, 1.9 μm particle size, L × I.D. 5 mm × 2.1 mm 3 ea 577133-U Titan C18 UHPLC Guard Cartridge Holder, 1.9 micron, for use with Titan Guard Cartridges 1 ea Analytical Standards of Alkylresorcinols 56453 5-Tridecylresorcinol 10 mg 91822 5-Pentadecylresorcinol 10 mg 97001 5-Heptadecylresorcinol 10 mg

49519 5-(Nonadecyl-1,1,2,2-d4)resorcinol 10 mg 57981 5-Nonadecylresorcinol 10 mg 53503 5-Eicosylresorcinol 10 mg 50851 5-Heneicosylresorcinol 10 mg 03422 5-Tricosylresorcinol 10 mg

sigma-aldrich.com/uhplc 16

LC-MS Ultra Solvents and Additives Designed for UHPLC Quality exceeding your expectations

Purity for low detection limits • Suitability tested by UHPLC-MS/TOF • Lot-to-lot reproducibility • Microfiltered (0.1 μm), filled in clear borosilicate glass containers For more information or to request a LC-MS Ultra CHROMASOLV Solvents

Chromatography and Additives Brochure (OUX), please visit sigma-aldrich.com/uhplc

GC-Headspace Solvents Description Size Cat. No. Specifically developed and optimized for sensitive static 1,3-Dimethyl-2-imidizolidinone 1 L 67484 GC-Headspace analysis of volatile organics. N,N-Dimethylacetamide 1 L 44901 Dimethyl sulfoxide 1 L 51779 ● High purity – microfiltered (0.2 μm) N,N-Dimethylformamide 1 L 51781 ● Improved analyte recoveries Water 1 L 53463 ● Longer shelf life – ed under inert gas GJMM 1-Methyl-2-pyrrolidinone, for GC-HS 1 L 69337 ● Specifications matching USP, Ph.Eur. & ICH guidelines Benzyl alcohol 1 L 80708 For additional information, visit us at sigma-aldrich.com/gc-hs.

sigma-aldrich.com/analytix 17

A Remarkable Carbonate-Selective Ionophore for the Determination of Oceanic Carbon Dioxide Using an Ion-Selective Electrode

Daniel Weibel, Product Manager Analytical Reagents [email protected]

Potentiometric detectors based on ion-selective electrodes Using 93206 as ionophore in the electrode, the total CO2 are used in a wide range of applications for detecting vari- level was measured in Yellow Sea water, providing a fast, ous ions important in the clinical, environmental and indus- stable, and reproducible potentiometric response. In this

trial areas because they offer advantages such as high manner, the level of total CO2 was determined to be selectivity, sensitivity, good precision, simplicity, portability, 1.94 ± 0.03 (n = 15) mmol/kg seawater, which was found to [1] non-destructive analysis, and low cost . be in good agreement to values determined by a CO2 gas sensor and a standard potentiometric titration method. To map out the global carbon cycle, it is very important to know detailed information about the spatial and temporal Recommended Membrane Composition (% by wt.) variations in inorganic carbons in oceans, especially in rela- Cat. No. Description wt% tion to the air-sea redistribution of the rapidly increasing 93206 Carbonate ionophore VII 5.1% anthropogenic carbon dioxide in the atmosphere and its 91661 Methyltridodecylammonium chloride 1.2% 02138 Bis(1-ethylhexyl)adipate 56.8% infl uence on the greenhouse eff ect[2]. 81392 Poly(vinyl chloride) 36.9% Dissolved in ethyl acetate and THF (3:5 v/v) A frequently measured parameter to quantify the oceanic Recommended Cell Assembly carbon system is the total CO2 in seawater. It is estimated from the total alkalinity determined by potentiometric Reference || sample solution || liquid membrane | 0.1 M NaH PO , 0.1 M Na HPO , 0.01 M NaC | AgCl, Ag titration or from the concentration of dissolved CO2 mea- 2 4 2 4 sured directly with coulometric methods, infrared spectroscopy, and gas chromatography, which, in seawater, is not Electrode Characteristics and Function Pot trivial and often requires rather elaborate instrumentation. Selectivity Coefficients log K Na,M as obtained by the matched potential method at pH 8.0 (0.1 M Tris-H SO ): Under normal seawater conditions, CO2(aq) is a minor species 2 4 2- (<1%), and the concentration of CO3 is about 10 –11% of the Pot -6.8 Pot -4.5 Pot -1.3 log K CO3,Cl : log K CO3,NO3 : log K CO3,Salicylate : total inorganic carbons. Hence, considering the relative Pot -6.7 Pot -4.8 log K CO3,Br : log K CO3,NO2 : 2- abundance of CO3 ion in seawater, carbonate-selective Pot -2.0 Pot -2.8 log K CO3,ClO : log K CO3,SCN : electrodes should be used in seawater analysis. Sensorics Slope: -26.0 mV/dec Nam et al. synthesized a new carbonate-selective iono- -7 2- Detection level: 5.8*10 mol/L CO3 phore (93206) that behaves like a molecular tweezer; two trifl uoroacetophenone groups were attached to a rigid cho- For more information on our sensoric applications, visit lic acid derivative (Figure 1). The observed carbonate selec- sigma-aldrich.com/selectophore Pot tivity of log K CO3,Cl = -6.8 of the electrode over chloride and other anions (incl. lipophilic ones) appeared to be high enough to apply to seawater analysis. CH CH3 3 O CH 3 N(C8H17)2 CH O 3 O CH O O 3 N(C8H17)2 F3COC H H C O 3 H O O O H H

O COCF3 H F3COC COCF3

Figure 1 Structural Representations of the Carbonate-Selective Ionophore 93206, Indicating the Tweezer-Like Shape of the Molecule on the Right.

References: [1] Selectophore® Ion Sensor Materials; Sigma-Aldrich Co.: St. Louis, MO, 2011. [2] Choi, Y. S.; Lvova, L.; Shin, J. H.; Oh, S. H.; Lee, C. S.; Kim, B. H.; Cha, G. S.; Nam, H. Determination of Oceanic Carbon Dioxide Using a Carbonate-Selective Electrode. Anal. Chem. 2002, 74, 2435–2440.

sigma-aldrich.com/selectophore 18

A New Approach for the Detection of Acidic Pesticides in Water by MS Increasing the Sensitivity in Pesticide Analysis by Paired-Ion ESI Detection

Jens Boertz, Product Manager Analytical Reagents [email protected] Rudolf Köhling, Sr. Scientist [email protected] Chengdong Xu, Graduate Student1, Zachary S. Breitbach, Research Scientist1, Daniel W. Armstrong, R.A. Welch Professor, 1Department of Chemistry & Biochemistry, University of Texas at Arlington [email protected]

In previous publications, di- and tri-cationic ion-pair In Table 1 also the results for 19 acidic pesticides using reagents were used to detect inorganic compounds, such three different ion-pairing reagents are presented. The lim- as chlorides and sulfates.[1-2]. A recent publication by C. Xu et its of detection (LOD) of 19 pesticides obtained were in the al [3] demonstrates the versatility of such ion-pair reagents, range from 0.6 to 19 pg. The fourth ion-pairing reagent which are added post-column to the mobile phase and applied (1,9-Nonanediyl-bis(3-methylimidazolium) difluo- increase the ESI-MS sensitivity extraordinarily by forming ride) did not show any improvement in terms of LOD. In positively charged adducts of reagent and anionic analytes total, this approach shows an increased sensitivity of one to (Figure 1). three orders of magnitude for the pesticides analyzed.

A novel method based on paired-ion electrospray ioniza- [Dicat I]2+ LC pump tion (PIESI) mass spectrometry has been developed for 40 µM paired [Analyte ion reagent + determination of acidic pesticides at ultratrace levels in in water + Dicat I] Mixing tee ESI-MS surface and ground waters. The proposed approach pro-

vides greatly enhanced sensitivity for acidic pesticides and overcomes the drawbacks of the less sensitive negative ion mode ESI-MS. The limits of detection (LODs) of 19 acidic Supelco Ascentis® Express column C18 pesticides were evaluated with four types of dicationic ion- Analyte™ Sample Solution pairing reagents (IPR) in both single-ion monitoring (SIM) and selected-reaction monitoring (SRM) mode. The LOD of MS pump 19 pesticides obtained with the use of the optimal dica- water/ methanol tionic ion-pairing reagent ranged from 0.6 pg to 19 pg, indicating the superior sensitivity provided by this method. Figure 1 The Instrumental Setup of HPLC-PIESI-MS. The transition pathways for different pesticide-IPR com- plexes during the collision-induced dissociation (CID) were Since the aim of water analysis is the detection of the small- identified. To evaluate and eliminate any matrix effects and est amounts of pesticides in water or other biological matri- further decrease the detection limits, off-line solid-phase ces to understand the effect on health and the environ- extraction (SPE) was performed for DI water and a river ment, this new method supports analytical laboratories in water matrix spiked with 2000 ng/L and 20 ng/L pesticide this challenge. standards respectively, which showed an average percent recovery of 93%. The chromatographic separation of the

Spectrometry LC-MS Method The different pesticides are separated on a Supelco Ascentis acidic pesticides was conducted by high-performance Express C18 (15 cm x 2.1 mm i.d., 2.7 µm particle size) column liquid chromatography (HPLC) using a C18 column using a gradient from 95% 5 mmol/L formic acid/water and (15 cm x 2.1 mm i.d., 2.7 µm particle size) in the reversed- 5% 5 mmol/L formic acid/methanol to 5% aqueous buffer phase mode using linear gradient elution. The optimized within 20 min at 0.4 mL/min. The ion-pair reagent is mixed HPLC–PIESI-MS/MS method was utilized for determination to the LC flow post-column via a mixing tee with a flow rate of acidic pesticides at ng/L level in stream/pond water of 0.1 mL/min. Injection volume of water samples is 25 µL samples. This experimental approach is one to three orders (fixed loop). This method demonstrates a very good sensi- of magnitude more sensitive for these analytes than other tivity with a standard LC-MS system. Table 1 shows the list of reported methods performed in the negative-ion mode. analytes analyzed with this technique.

sigma-aldrich.com/ipc 19

Sample 1,5-Pentanediyl-bis(1-butylpyrrolidinium) 1,3-Propanediyl-bis(tripropylphosphonium) 1,5-Pentanediyl-bis(3-benzylimidazolium) difluoride difluoride difluoride SIM SRM SIM SRM SIM SRM LOD (pg) m/za LOD (pg) m/zb LOD (pg) m/za LOD (pg) m/zb LOD (pg) m/za LOD (pg) m/zb 2,4-D 45 543.3 7.0 416.2 10 581.3 8.0 187.2 16 605.2 8.0 385.3 MCPA 3.6 523.4 1.1 396.3 15 561.3 15 187.2 10 585.3 10 385.3 2,4,5-T 25 577.3 3.7 450.2 16 615.3 24 187.2 25 639.2 3.7 385.3 2,4-DB 37 571.4 6.7 444.3 700 609.3 700 187.2 2000 633.3 2000 385.3 MCPB 30 551.4 9.0 424.3 1100 589.4 720 187.2 1300 613.3 1200 385.3 2,4,5-TB 32 605.3 6.0 478.3 1800 643.3 6000 187.2 5300 667.2 2500 385.3 Cloprop 9.0 523.4 2.7 396.3 10 561.3 10 187.2 7.0 585.3 3.5 385.3 Dichlorprop 30 557.3 4.5 430.3 20 595.3 12 187.2 10 619.2 4.0 385.3 Fenoprop 20 591.3 4.0 464.3 20 629.3 20 187.2 30 653.2 10 385.3 Mecoprop 30 537.4 12 410.3 3.8 575.4 2.8 187.2 15 599.3 4.7 385.3 Dicamba 15 543.3 3.0 416.3 6.5 581.3 6.5 187.2 6.0 605.2 2.6 385.3 2,3,6-TBA 110 547.3 6.3 420.2 60 585.2 60 187.2 17 609.2 3.8 385.3 Clopyralid 20 514.3 1.5 387.2 4.5 552.3 4.5 187.2 5.0 576.2 5.0 385.3 Quinclorac 6.0 564.3 0.60 437.2 7.5 602.3 7.5 187.2 10 626.2 3.0 385.3 Quinmerac 15 544.4 2.8 417.3 7.0 582.3 5.2 187.2 3.5 606.3 3.5 385.3 Flupropanate 5.0 469.4 5.0 342.2 3.5 507.3 1.1 187.2 3.1 531.3 0.088 385.3 MCA 6.0 417.3 3.0 290.2 10 455.3 35 187.2 17 479.2 15 385.3 TCA 160 485.2 19 359.3 1.5 523.2 2.0 187.2 15 547.1 7.5 385.3 Dalapon 10 465.3 0.75 359.3 5.0 503.3 5.0 187.2 15 527.2 2.9 385.3

Table 1 Limits of Detection of Pesticide Standard Solutions Obtained by PIESI-MS in the SIM Mode and SRM Mode with the Use of Dicationic Ion-Pairing Reagent. a Indicates the mass-to-charge ratio of the complex monitored in the SIM mode. b Indicates the mass-to-charge-ratio of the SRM fragment monitored in the SRM mode.

Cat. No. Name (Abbreviation) Exact Mass Package Size 31518 2,4-dichlorophenoxyacetic acid (2,4-D) 219.97 250 mg 45555 4-chloro-o-tolyloxyacetic acid (MCPA) 200.02 250 mg 45667 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) 253.93 250 mg 45420 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) 248.00 250 mg 36145 4-(4-chloro-o-tolyloxy)butyric acid (MCPB) 228.06 100 mg S412325 4-(2,4,5-trichlorophenoxy)butyric acid (2,4,5-TB) 281.96 25 mg 233013 2-(3-chlorophenoxy)propionic acid (cloprop) 200.02 50 g 45436 2-(2,4-dichlorophenoxy)propionic acid (dichlorprop) 233.99 250 mg 45691 2-(2,4,5-trichlorophenoxy)propionic acid (fenoprop) 267.95 250 mg 36147 2-(4-chloro-o-tolyloxy)propionic acid (mecoprop) 214.04 100 mg 45430 3,6-dichloro-2-methoxy-benzoic acid (dicamba) 219.97 250 mg R169676 2,3,6-trichlorobenzoic acid (2,3,6-TBA) 223.92 25 mg

36758 3,6-dichloro-2-pyridine-carboxylic acid (Clopyralid) 190.95 250 mg Spectrometry 36521 3,7-dichloro-8-quinoline-carboxylic acid (Quinclorac) 240.97 250 mg 36522 7-chloro-3-methyl-8-quinoline-carboxylic acid (Quinmerac) 221.02 250 mg 402923 2-chloroacetic acid (MCA) 93.98 100 g T6399 2,2,2-trichloroacetic acid (TCA) 161.90 5 g 35562 2,2-Dichloropropionic acid (dalapon) 141.96 250 mg 56618 1,5-Pentanediyl-bis(1-butylpyrrolidinium) difluoride solution 100 mL 75128 1,9-Nonanediyl-bis(3-methylimidazolium) difluoride solution 100 mL References 42341 1,3-Propanediyl-bis(tripropylphosphonium) difluoride solution 100 mL [1] Köhling, R.; Reichlin, N.; Wille, G. Analytix No. 9. 76507 1,5-Pentanediyl-bis(3-benzylimidazolium) difluoride solution 100 mL [2] Köhling, R.; Reichlin, N. 53802 Supelco Ascentis Express ™ C18 (15 cm x 2.1 mm i.d., 1 EA Analytix No. 3. 2.7 μm particle size) [3] Chengdong X.; Armstrong, D. W. 94318 Formic acid 50 mL, 250 mL Analytica Chimica Acta, 792. 2013 1–9. 34485 Methanol 2.5 L, 4x2.5 L Table 2 Chemicals Used to Carry Out HPLC-PIESI-MS Experiments .

sigma-aldrich.com/ipc 20

Are you familiar with our UV-Vis optical components line from Hellma®? Check out our website at sigma-aldrich.com/hellma Our portfolio of Hellma products includes: • Cells for absorption, fluorescence and special application measurements • UV-Vis reference materials • Cleaning concentrate for cells and optical components

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• Produced under double accreditation, fulfilling ISO/IEC 17025 and ISO Guide 34 • The certified value of the reference material is directly traceable to the SI unit (kg) and is also measured against an internationally recognized reference material (from NIST, BAM or other) • Information about traceability and uncertainty are included in the certificate according to ISO Guide 31, and expiry date is also given • Certificates can be accessed on the Web by using the product and lot numbers • IC standards offered in HDPE bottles • In most cases, relevant impurities are stated for the inorganic single-ion standards Visit our website to view IC high-purity Certifi ed Reference Standards at: sigma-aldrich.com/ic

sigma-aldrich.com/analytix 21

Water Determination in Pharmaceutical Compounds Karl Fischer Titration with HYDRANAL® Reagents

Andrea Felgner, Market Segment Manager [email protected]

Water-content determination is mandatory for many mate- according to standard procedures. 5-Aminolevulinic acid- rials used in the manufacturing of medicines. Karl Fischer HCl (lyophilisate) shows a strong side reaction in methanol; (KF) titration is the long-standing standard method for this an end point cannot be reached with direct KF titration analysis prescribed by the leading pharmacopoeias, such as methods. For this compound, we recommend determina- the European (Ph.Eur.), the United States (USP) and the Japa- tion with the KF oven in combination with the coulometric nese (JP). titration technique, due to the low water content of this substance. Suitable application parameters are (application Volumetric Karl Fischer Technique L506): The volumetric KF technique can be used for samples with • Evaporation temperature 80 °C (decomposition of the high water content (approx. 0.1–100%). The sample is added sample starts at 120 °C) to the titration vessel containing a suitable working medium, • Determination time 600 seconds such as Hydranal Methanol dry or Hydranal Solvent. With • Anolyte Hydranal Coulomat AG or Hydranal Coulomat the corresponding iodine-containing titrating agent, AG-Oven Hydranal Composite or Hydranal Titrant, the water content • Catholyte Hydranal Coulomat CG of the sample is determined by titration. End-point determination is carried out by applying a constant current and Side Reactions and pH-Influencing Samples measuring the voltage via double platinum electrode; or Substances containing nitrogen compounds may cause vice versa, applying a constant voltage and measuring the interference with the pH value of the working medium. current. The water content is calculated from sample Side reactions occur during the KF titration, possibly lead- weight, consumption of titrating agent, and water equiva- ing to coated electrodes, fading end points or no end lent (titer) of the titrating agent. Pharmaceutical com- points at all, and erroneous results. These side reactions can pounds such as ethosuximide, used for epilepsy treatment, be suppressed through the addition of suitable buff er sub- (application L510), or the cytostatic drug cyclophospha- stances to the working medium in the titration vessel. mide (application L463), can be analyzed according to standard procedures without interference. These titrations can Benserazide hydrochloride, an active ingredient used in the also be carried out in less toxic ethanol-based reagents, the treatment of Parkinson’s disease, is an example of a nitro- Hydranal E-types. gen-containing substance. For correct determination of its water content, Hydranal Buff er Base or a mixture of Hydranal Coulometric Karl Fischer Technique Methanol dry and salicylic acid should be used as a working Samples with lower water content (up to 1%) can be ana- medium, and then be titrated with Hydranal Composite 2 lyzed using the coulometric KF method. The required (application L416). Profl avine hemisulfate, a topical antisep- iodine is generated electrochemically in the titration vessel tic, also increases the pH value of the working medium and by anodic oxidation of iodide, which is contained in the no end point is achieved. Hydranal Buff er Base or a mixture Hydranal Coulomat reagents. The water content is calcu- of Hydranal Methanol dry and benzoic acid can be used to lated from the amount of the produced (consumed) elec- lower the pH value and prevent the side reaction; titrate tric current (which equals iodine consumption) over time. with Hydranal Composite 5 (application L354). Water-con-

tent determination in penicillin can also be disturbed by pH Titration Karl Fischer Oven Technique infl uences; a side reaction occurs: penicillin derivatives such Insoluble samples, samples that undergo side reactions as penicilloic acid and other hydrolysis products are oxi- with the KF reagents, or samples that release their water dized by iodine. By conducting the titration in weakly acidic only at high temperatures, may be analyzed using the KF conditions, this side reaction can be suppressed. The KF oven. The water of the sample is driven out at a variable one-component technique with Hydranal Methanol dry or temperature and transferred by a suitable carrier gas into Hydranal Methanol Rapid and Hydranal Composite 2 gives the KF titration vessel. The water content is determined a pH value of approximately 5 in the titration vessel. This is

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suffi cient for titrating the water content in penicillin with- Application Reports out any side reactions. If an end point is not easily reached, All application reports can be obtained by contacting our addition of salicylic acid to the working medium (before Hydranal technical service team ([email protected]). Visit pre-titration) is recommended. Titration of penicillin with our Web site sigma-aldrich.com/hydranal for complete the KF two-component technique is also possible. A mix- product listings and more information on KF titration. ture of Hydranal Solvent and salicylic acid is recommended as a working medium and can be titrated with Hydranal On our Web site sigma-aldrich.com/hydranal you will Titrant (application L166). fi nd more information about the Ph. Eur. requirements for KF titration. Our Hydranal technical service team has carried Enhancement of Sample Solubility out suitability tests for a range of substances, which are For samples that are only poorly soluble in the KF working available upon request. medium, Hydranal-Buffer Base or a mixture of Hydranal Methanol dry and salicylic acid can be recommended as Take advantage of both our expertise gained from over working media to enhance solubility and therefore yield thirty years of experience and our extensive applications correct determination results (titrating agent Hydranal database on KF titration. For any questions, help, or feed- Composite). Examples are the beta-lactam antibiotics back, please visit our Web site or contact us (details below). amoxycillin-3-hydrate (application L352) or ampicillin (application L422).

However, other substances such as riboflavin phosphate sodium (biochemical cofactor, also used as a food dye) prove insoluble in the alcohol-based KF media. As a suitable solubilizer for this compound, formamide is recommended. It can be added to the working medium (volumetric KF technique) in a ratio of 1:1 (application L509).

Cat. No. Description L166 Penicillin-G sodium and penicillin-G-potassium L230 Dobutamine hydrochloride L242 Erythromycin L249 Adenosine-5’-triphosphoric acid disodium salt L297 Glycerine monostearate L307 Benzyl penicillin procaine L326 Magnesium stearate L352 Amoxycillin-3-hydrate L354 Proflavine hemisulfate L416 Benserazide hydrochloride L422 Ampicillin L448 Gentamicin sulfate L463 Cyclophosphamide L489 Disodium hydrogen phosphate-12-hydrate L506 Aminolevulinic acid-HCl (Lyophylisate) L509 Riboflavin phosphate sodium L510 Ethosuximide L604 Povidon

Titration L607 Sorbitol solution (70%) L608 Polysorbate 80 L660 Ibuprofen (film tablets) L661 Zineb L664 Niclosamide hydrate L670 Atrazine desisopropyl L675 Atrazine desisopropyl-2-hydroxy L698 Asulam

Table 1 Selected Application Reports for the Pharmaceutical Figure 1 Application Report for Polysorbate 80 (L608) Industry

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Hydranal Technical Helpline for expert support and customer service:

Europe and International USA and Canada Mr. Thomas Wendt Mr. Doug Clark Technical Service Hydranal Hydranal Technical Center Wunstorfer Straße 40 545 S. Ewing Ave D-30926 Seelze, Germany St. Louis MO 63103, USA Tel. +49 5137 8238-353 Toll free: +1 800 493-7262 Fax +49 5137 8238-698 (USA and Canada) E-mail: [email protected] Fax: +1 314 286-6699 E-mail: [email protected]

Cat. No. Description Reagents for Volumetric KF Titration 34805 Hydranal Composite 5, reagent for one-component KF titration 34806 Hydranal Composite 2, reagent for one-component KF titration 37817 Hydranal Methanol Rapid, working medium for fast one-component KF titration 34741 Hydranal Methanol dry, working medium for one-component KF titration 34801 Hydranal Titrant 5, reagent for two-component KF titration 34811 Hydranal Titrant 2, reagent for two-component KF titration 34800 Hydranal Solvent, working medium for two-component KF titration 34724 Hydranal Formamide dry, solvent for KF titration 34804 Hydranal Buffer Acid, buffer substance for KF titration 37859 Hydranal Buffer Base, buffer substance for KF titration 37865 Hydranal Salicylic Acid, buffer substance for KF titration 32035 Hydranal Benzoic Acid, buffer substance for KF titration Reagents for Coulometric KF Titration 34836 Hydranal Coulomat AG, anolyte reagent 34739 Hydranal Coulomat AG-Oven, anolyte reagent for KF oven 34840 Hydranal Coulomat CG, catholyte reagent for cells with diaphragm Water Standards for KF Titration 34849 Hydranal Water Standard 10.0, 1 g contains 10.0 mg = 1.0% water, tested against NIST SRM 2890 (exact value on CoA) 34828 Hydranal Water Standard 1.0, 1 g contains 1.0 mg = 0.1% water, tested against NIST SRM 2890 (exact value on CoA 34847 Hydranal Water Standard 0.1, 1 g contains 0.1 mg = 0.01% water, tested against NIST SRM 2890 (exact value on CoA) 34696 Hydranal Standard sodium tartrate dihydrate, solid standard for volumetric KF titration, water content ~15.6% (exact value on CoA) Table 2 Selected Hydranal Reagents for KF titration

Request the new Hydranal Product Line Overview brochure • Volumetric Titration with One-Component Reagents • Volumetric Titration with Two-Component Reagents • Coulometric Titration with Hydranal Coulomat Titration • Hydranal Water Standards • Hydranal E-Type Reagents • Reagents for Ketones and Aldehydes • Technical Support

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