	Generally speaking, chromate - Cr(VI) - is classified as allergenic, carcinogenic, and extremely toxic and is subject to strict monitoring. Cr(VI) can occur in various concentrations in different areas, for example, drinking water, toys, or textile and leather products. Metrohm has developed ion chromatographic determination methods for determining Cr(VI) in various concentration ranges (ng/L to mg/L) with inline sample preparation techniques for various matrices. The method can be almost entirely automated. In the following application note, we present the analysis of hexavalent chromium in toys, leather, and drinking water.

Chromate in Toys According to DIN EN 71-3

	Children are exposed to heavy metals from a wide variety of sources. Cr(VI) in particular represents a potential hazard because it is absorbed from food and drinking water, from the air, from textiles, and from toys. Analytical determination of the Cr(VI) content in toys is described in the European Standard DIN EN-71-3-2013 (Safety of toys Part 3 - Migration of certain elements), as well as in the EU directive 2009/48/EC. The limit values that apply to Cr(VI) according to the EU directive are listed in Table 1.

	According to DIN EN 71 Part 3, the user is free to choose whichever analysis method he or she prefers, as long it is validated. Metrohm has developed an ion chromatography method for this application that uses Inline Preconcentration and Inline Matrix Elimination. The method can be almost entirely automated.

Sample Digestion Using Synthetic Gastric Juice

	Chromium(VI) is extracted from the toy material at body temperature using hydrochloric acid. This type of sample digestion simulates how gastric juice dissolves out the harmful substance from swallowed toy material. The sample that is obtained using this method is then manually neutralized and diluted. Dilution is required because the high ion concentration present in the sample - which is caused by hydrochloric acid extraction and subsequent neutralization - does not permit preconcentration of chromium(VI).

Automation Improves Convenience and Safety

	All the remaining steps are automated. To start with, the entire sample flow path is equilibrated using sample. A Dosino then feeds a defined sample volume onto the preconcentration column (Figure 1). The Dosino can accurately control the injection volume, and this is what forms the basis of reliable determination, especially when dealing with low analyte concentrations. Equally important for the sensitivity, however, is the post-column reaction of Cr(VI) with 1,5-diphenylcarabazide that results in the formation of VIS active complexes. As illustrated in Figure 2, the method presented accurately determines hexavalent chromium in the single digit ppt range, easily complying with the 10 ppt limit set by the EU directive 2009/48/EC.

Chromate in Leather According to DIN EN ISO 17075

	The Cr(VI) that may form during the chrome tanning process is regarded as allergenic and carcinogenic. Causes of the Cr(VI) load in leather include, among other things, contamination of the Cr(III) salts by Cr(VI) compounds. In addition, excess Cr(III) salts that are not bound to the collagen in the skin of the leather may oxidize to form chromium(VI) under certain conditions.

	The Cr(VI) content in leather is determined in accordance with DIN EN ISO 17075:2007. Strongly coloured leather extracts disrupt the determination of chromium(VI), and require filtration of the extracts.

	Metrohm has developed an ion chromatography method for this application using Inline Dialysis for automatic sample preparation and UV–vis detection following post-column reaction. Before injection of the sample on the separation column, the highâmolecular compounds of the sample matrix are separated from Cr(VI) with Inline Dialysis. In the second step, chromatographic separation takes place on an anion exchanger before post-column reaction with 1,5-diphenylcarbazide, as already described in the toy section.

	Advantages of the method in comparison with the photometric method with solid-phase extraction include:

	The moderate price of the membrane for Inline Dialysis keeps operating costs very low in comparison with solid-phase extraction.

	The automatic sample preparation of the next sample is already taking place with Inline Dialysis during the running time of the chromatogram of each sample.

	Detection sensitivity can be enhanced considerably in comparison with the conventional photometric method thanks to the sensitivity of the UV–vis detector after post-column reaction.

Chromate in Mineral and Drinking Water According to EPA 218.7

	Cr(VI) is a highly toxic contaminant of drinking water. Even the smallest quantities are hazardous to human health.

	Even though the RoHS Directive has severely restricted the use of Cr(VI) compounds in Europe since 1 July 2006, chromate nonetheless remains a widespread pollutant in the environment.

	Some pigments formerly used in dyes also contained Cr(VI) compounds. These chromate pollutants appear in the environment when decontamination is done improperly. The carcinogenic Cr(VI) can then find its way into the soil, and over time also into our water.

	The EU limit value for total chromium, which corresponds to both the WHO limit value and to the German Drinking Water Ordinance, is still set at 50 μg/L. There are ongoing discussions as to whether this limit value should be considerably lowered.

	Metrohm has developed an exceptionally sensitive ion chromatography method for the detection of Cr(VI), with a limit of determination of less than 0.02 μg/L chromate (Figure 3). The application is also in compliance with the limit value stipulated in EPA Standard 218.7.

	If required, the sample can be prepared for injection using Inline Ultrafiltration in an automated sequence. As described earlier, detection takes place by post-column reaction with 1,5-diphenylcarbazide and subsequent detection at 540 nm. The procedure is controlled using the intelligent ion chromatography software MagIC Net.

		DIN EN 71-3:2013-07: Safety of toys - Part 3: Migration of certain elements.	

		U.S. Environmental Protection Agency, Method 218.7, Version 1.0, Cincinnati, Ohio, USA (2011).	

		Metrohm Whitepaper WP-001EN: Chromium(VI) determination in children’s toys.	

		Metrohm Application Note AN-U-015: Chromium(VI) in a leather extract.	

		Metrohm Application Note AN-U-057: Chromate in drinking water by ion chromatography with PCR and UV–vis detection, according to EPA Method 218.7.

Articles

Environmental

Chromate in Toys, Leather, and Potable Water

	Neonicotinoids are a class of insecticides widely used in agriculture to protect fields (corn, canola, soybeans) as well as fruits and vegetables. As contact or stomach insecticides, they are effective mostly against sucking and some chewing insects. Neonicotinoids are neuro-active insecticides and have an impact on the central nervous system of insects causing paralysis and death.

	The first neonicotinoid, imidacloprid, was patented by Bayer CropScience. The systemic distribution of neonicotinoids throughout the whole plant has high efficacy against sucking insects. The long residual activity made these insecticides very popular within the global pesticide market.

	Today, there are about 10 molecules classified as neonicotinoids; these include acetamiprid, clothianidin, imidacloprid, thiacloprid, thiamethoxam, dinotefuran, and nitempyram. These active components are commercialized under various brand names by international companies.

	Recently, the use of these compounds has become controversial because they have been singled out as one cause of the honeybees’ Colony Collapse Disorder (CCD). Since pollination is essential for agriculture, extensive studies have been conducted to evaluate the impact of neonicotinoids on bee health.

	The European Food Safety Authority (EFSA) has identified risks to bees in the use of neonicotinoids. Three types of insecticides are concerned: clothianidin, imidacloprid, and thiamethoxam. They may have acute and chronic effects on survival and development of bee colonies, their behaviour, and their larvae.

	Following this, the EFSA limited the use of thiamethoxam, clothianidin, and imidacloprid. Some European countries have banned or restricted the use of neonicotinoids.

	In order to better understand the effect of these compounds on bees and their contamination in pollen and honey, a highly sensitive assay method is required. For that purpose, an ultrahigh-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS–MS) analysis was developed.

 All the analytical standards were provided by Sigma-Aldrich. The internal standards thiamethoxam-d3, imidacloprid-d4, and chlothianidin-d3 were purchased from SigmaâAldrich. The solvents used, including water and mobile phase additive, were of UHPLC–MS quality (Biosolve).

 Compound extraction was performed using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method with an additional dispersive solid-phase extraction (dSPE) step.

	A sample of 5 g of honey (± 1%) was weighted in a 50 mL polypropylene tube. Five microlitres of internal standard solution at 5 µg/mL of each compound in acetonitrile was added to the honey and was dried for 10 min. Ten millilitres of ultrapure water were added and the samples were homogenized by vortex mixing for 1 min. Ten millilitres of acetonitrile were then added followed by vortex mixing for 1 min.

	A salts mix (4 g MgSO4, 1 g sodium citrate, 0.5 g sodium citrate sesquihydrate, 1 g NaCl; Biotage Q0020-15V) was added to the samples. After manual shaking, samples were centrifuged at 3000 g for 5 min at 10 ºC.

	The supernatant (6 mL) was transferred into a 15 mL tube containing 1200 mg of MgSO4, 400 mg PSA, and 400 mg C18 (Biotage Q0050-15V). After centrifugation at 3000 g and 10 ºC for 5 min, the supernatant was transferred into inert glass vials for analysis (Shimadzu LabTotal 227-34001-01).

 Analysis was performed using a Nexera X2 UHPLC system coupled with a LCMS-8060 triple quadrupole mass spectrometer with heated ESI in positive ionization. All parameters are described in Tables 1 and 2.

	Mobile phase composition was optimized to generate the highest sensitivity. Ion source parameters (gas flows, temperatures) were also optimized using the Interface Setting Support Software (Shimadzu Corp.)

	As the final extract is in an organic solvent, large injection volumes are not possible without compromising separation and peak shapes. Evaporating the extracts and reconstituting them in aqueous solvent is tedious and generates additional costs as well as the risk of losses. Therefore, a special injection mode POISe (Performance Optimized Injection Sequence) was used that enables co-injection of water with the sample to counteract the organic solvent effect.

	Calibration: Calibration curves were prepared in acetonitrile to obtain final concentrations ranging from 2.5 pg/mL (2.5 fg on column) to 5 ng/mL. These concentrations correspond to 5 ppt and 10 ppb in honey, respectively. A typical calibration curve is shown in Figure 1.

	Recovery: Eight different honeys from the local supermarket were extracted with and without spike at 0.1 ppb. Each extract was injected five times to evaluate repeatability in matrix samples. A blank extract (no honey) was prepared to evaluate losses or non-specific interactions. Results are presented in Table 3.

	Real Sample Analysis: The eight samples used for recovery experiments were then assayed as unknowns. Thanks to the very high sensitivity reached, even low concentrations of neonicotinoids were quantified. The detailed results are presented in Table 4.

	A method for the ultra-sensitive assay of neonicotinoids in honey was set up. Sample preparation was simple but provided excellent recoveries, regardless of the honey type. The injection mode used prevented the use of tedious evaporation/reconstitution or dilution steps. The sensitivity obtained enabled an assay in real samples at very low levels far below the regulated residue levels. This method can be a very efficient support tool to better understand the impact of neonicotinoids on honeybee colonies.

Albert-Hahn-Str. 6–10, D-47269 Duisburg, Germany

Food and Beverage

The Impact of Neonicotinoids on Honeybee Colonies: Ultra-Sensitive and Rapid Assay of Neonicotinoids in Honey by UHPLC–MS–MS

	Benzodiazepines are psychoactive drugs widely prescribed for treating anxiety, insomnia, agitation, seizures, muscle spasms, and alcohol withdrawal. Common sample preparation methods to monitor for this drug class in whole blood include a protein precipitation step followed by either a time-consuming
	liquid–liquid extraction (LLE) or solid-phase extraction (SPE) procedure. This featured application note describes an easy, fast, and effective method using the QuEChERS approach to provide for their quantitative analysis.

		Add 2 mL of acetonitrile with 0.4% formic acid to a 15 mL centrifuge tube containing 400 mg MgSO4 and 100 mg NaOAc.

		Add internal standards (IS), and appropriate amounts of benzo spiking solution to fortified samples.

		Add 1 mL of the negative whole blood into the 15-mL tubes.

		Cap and shake for 1 min at 1000 strokes/min using a Spex 2010 Geno-Grinder.

		Transfer 1 mL of the supernatant to a 2-mL dSPE tube containing 150 mg MgSO4 and 50 mg PSA and 50 mg C18.

		Shake 1 min at 1000 strokes/min using the Spex 2010 GenoâGrinder.

		Transfer 0.4 mL of the cleaned extract into a 2-mL auto-sampler vial, add 0.4 mL of reagent water, and vortex for 30 s.

		The samples are now ready for LC–MS–MS analysis.

         Agilent 1200 Binary Pump SL / AB Sciex API 4000 Qtrap MS–MS

               100 × 2.1 mm, 3-μm

	                      UCT Selectra® DA LC column,

	            UCT Selectra® DA guard column,

	Excellent recoveries (85.5–105%) and relative standard deviations (RSD% ≤ 10.7%) were achieved. Matrix effects were found to be minor, ranging from -22% to 18%. This indicates that the QuEChERS technique with dSPE cleanup quickly and effectively removes unwanted matrix interferences that may cause significant ion suppression or enhancement.

2731 Bartram Road, Bristol, Pennsylvania 19007, USA

Medical/Biological

Quantitative Analysis of Benzodiazepines in Whole Blood by QuEChERS and LC–MS–MS

	There has been a significant resurgence in the development of antibody-drug conjugates (ADC) as target-directed therapeutic agents for cancer treatment. Among the factors critical to effective ADC design is the Drug Antibody Ratio (DAR). The DAR describes the degree of drug addition that directly impacts both potency and potential toxicity of the therapeutic, and can have significant effects on properties such as stability and aggregation. Determination of DAR is, therefore, of critical importance in the development of novel ADC therapeutics.

	DAR is typically assessed by mass spectrometry (MALDI–TOF or ESI–MS) or UV spectroscopy. Calculations based on UV absorption are often complicated by similarities in extinction coefficients of the antibody and small molecule. Mass spectrometry, though a powerful tool for Mw determination, depends on uniform ionization and recovery between compounds - which is not always the case for ADCs.

	Here we present a method for DAR determination based on size-exclusion chromatography combined with multi-angle light scattering (SEC–MALS) in conjunction with UV absorption and differential refractive index detection. Figure 1 shows UV traces for two model ADCs; molecular weights of the entire ADC complexes are determined directly from light scattering data.

	Component analysis is automated within the ASTRA 6 software package by using the differential refractive index increments (dn/dc) and extinction coefficients, which are empirically determined for each species or mined from the literature, to calculate the molar mass of the entire complex as well as for each component of the complex.

	In this example an antibody has been alkylated with a compound having a nominal molecular weight of 1250 Da (Figure 2). Molar masses of the antibody fractions are similar, which indicates that the overall differences between the two formulations reflect distinct average DARs that are consistent with values obtained by orthogonal techniques. Note that the molar mass traces for the conjugated moiety represent the total amount of attached pendant groups; the horizontal trends indicate that modification is uniform throughout the population eluting in that peak.

6300 Hollister Avenue, Santa Barbara, California 93117, USA

+1 (805) 681 9009  fax: +1 (805) 681 0123

The Application Notebook-03-01-2016

Chromate in Toys, Leather, and Potable Water

The Impact of Neonicotinoids on Honeybee Colonies: Ultra-Sensitive and Rapid Assay of Neonicotinoids in Honey by UHPLC–MS–MS

Quantitative Analysis of Benzodiazepines in Whole Blood by QuEChERS and LC–MS–MS

Antibody Drug Conjugate (ADC) Analysis with SEC–MALS