Determination of butyltin compounds in environmental samples by isotope-dilution GC–ICP–MS

Isotope-dilution analysis in combination with GC-ICP-MS detection has been applied to the determination of butyltin species in environmental samples. Different spikes containing the isotopically labeled butyltin species have been synthesized in the laboratory after optimization of the reaction conditions. The isotopic compositions of the tin species in the different spike solutions were determined by GC-ICP-MS after derivatization by aqueous ethylation with sodium tetraethylborate. Reverse isotope-dilution analysis was used for quantitation of the spike solutions by means of natural MBT, DBT, and TBT standards. The mixed spikes were used for simultaneous analysis of MBT, DBT and TBT in the certified reference materials, PACS-2, CRM 462, and CRM 646, with satisfactory results. The excellent agreement of the different speciation results obtained by use of the different spikes is a good indicator of the precision, accuracy, and reliability which can be achieved by using isotope-dilution analysis for trace metal speciation.Application of a double spike containing (119)Sn-enriched MBT (79.7 At%), (118)Sn-enriched DBT (86.7 At%), and (119)Sn-enriched TBT (83.1 At%) also enabled evaluation of the conditions resulting in quantitative extraction of the species from the solid matrix, in combination with possible alterations depending on the different extraction procedures used (mechanical shaking, ultrasounds, and microwaves). Mathematical equations used for this purpose computed the correct species concentrations directly and, additionally, the decomposition factors (from TBT to DBT and from DBT to MBT) after precise measurement of the (119)Sn/(120)Sn and (118)Sn/(120)Sn ratios for all butyltin species by GC-ICP-MS.     

Accurate determination of sulfur in gasoline and related fuel samples using isotope dilution ICP–MS with direct sample injection and microwave-assisted digestion

Inductively coupled plasma isotope-dilution mass spectrometry (ICP–IDMS) with direct injection of isotope-diluted samples into the plasma, using a direct injection high-efficiency nebulizer (DIHEN), was applied for accurate sulfur determinations in sulfur-free premium gasoline, gas oil, diesel fuel, and heating oil. For direct injection a micro-emulsion consisting of the corresponding organic sample and an aqueous ³⁴S-enriched spike solution with additions of tetrahydronaphthalene and Triton X-100, was prepared. The ICP–MS parameters were optimized with respect to high sulfur ion intensities, low mass-bias values, and high precision of ³²S/³⁴S ratio measurements. For validation of the DIHEN–ICP–IDMS method two certified gas oil reference materials (BCR 107 and BCR 672) were analyzed. For comparison a wet-chemical ICP–IDMS method was applied with microwave-assisted digestion using decomposition of samples in a closed quartz vessel inserted into a normal microwave system. The results from both ICP–IDMS methods agree well with the certified values of the reference materials and also with each other for analyses of other samples. However, the standard deviation of DIHEN–ICP–IDMS was about a factor of two higher (5–6% RSD at concentration levels above 100 g g^–1) compared with those of wet-chemical ICP–IDMS, mainly due to inhomogeneities of the micro-emulsion, which causes additional plasma instabilities. Detection limits of 4 and 18 g g^–1 were obtained for ICP–IDMS in connection with microwave-assisted digestion and DIHEN–ICP–IDMS, respectively, with a sulfur background of the used Milli-Q water as the main limiting factor for both methods.This paper was presented as a poster at the 2004 winter conference on plasma spectrochemistry, Fort Lauderdale, January 5–10, 2004     

Comparison between GC–MS and GC–ICPMS using isotope dilution for the simultaneous monitoring of inorganic and methyl mercury,butyl and phenyl tin compounds in biological tissues

The aim of this work is to compare simultaneous isotope dilution analysis of organotin and organomercury compounds by gas chromatography–mass spectrometry (GC–MS) and gas chromatography–inductively coupled plasma mass spectrometry (GC–ICP/MS) on certified bivalve samples. These samples were extracted by microwave with tetramethylammonium hydroxide (TMAH). Derivatization with both NaBEt₄ and NaBPr₄ was evaluated, and analytical performances were compared. Two CRM materials, BCR-710 and CRM-477, were analyzed by both techniques to verify accuracy. A mixed spike containing ²⁰¹Hg-enriched methylmercury (MeHg), ¹⁹⁹Hg-enriched inorganic mercury (iHg), ¹¹⁹Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) as well as homemade ¹¹⁶Sn-enriched monophenyltin (MPT), diphenyltin (DPT), and triphenyltin (TPT) was used for the isotope dilution analysis of samples. The two techniques studied were compared in terms of classic analytical parameters: linearity, precision or repeatability (i.e., percent relative standard deviation, RSD%), limit of detection (LOD), and limit of quantification (LOQ), showing excellent linearity, precision below 12 % for all analytes, and LOQs of 0.06–1.45 pg for GC–MS and 0.02–0.27 pg for GC–ICP/MS.

Simultaneous determination of five synthetic antioxidants in edible vegetable oil by GC–MS

A simple, quick and nontoxic analytical method for the simultaneous determination of five synthetic antioxidants [t-butyl-4-hydroxyanisole (BHA), 2,6-di-t-butyl-hydroxytoluene (BHT), t-butyl hydroquinone (TBHQ), ethoxyquin (EQ) and 2,6-di-tert-butyl-4-hydroxymethyl-phenol (Ionox 100)] in edible vegetable oil has been developed. The analytes were extracted by ethanol, then separated and detected by GC–MS. Extraction conditions such as volume of ethanol required, mixing time and number of extractions were investigated and optimized by an orthogonal array experimental design. The five compounds behaved linearly in the 0.100∼20.0 mg/L concentration range, and the limits of detection (LOD) for BHA, BHT, TBHQ, EQ and Ionox-100 were 1.00, 0.92, 11.5, 0.83 and 1.39 μg/L, respectively. The recoveries at the tested concentrations of 1.00, 20.0 and 100 mg/kg were 75.6∼123%, with coefficients of variation <10.0%. The proposed procedure was successfully applied to the simultaneous analysis of the five antioxidants in soybean oil, tea oil, edible blended oil, rap oil, peanut oil, peanut blended oil and sesame oil samples purchased from local supermarkets.     

Application of isotope dilution ICP–MS techniques to quantitative proteomics

ICP–MS techniques based on isotope dilution analysis can be regarded as an emerging tool in quantitative protein analysis. Well-known concepts, for example species-specific and unspecific isotope dilution analysis, which promoted accurate and precise quantification in elemental speciation studies, have nowadays been transferred to the analysis of large biomolecules, e.g. proteins. Besides detection of heteroatom-containing proteins, the artificial introduction of metal-containing labels has attracted much attention and, as a consequence, ICP–MS-based isotope dilution techniques can serve as a valuable quantification tool. In particular, because isotope dilution ICP–MS techniques can enable absolute protein quantification, they can be regarded as an attractive technique in current and prospective proteomics. In this review, recent developments and applications will be highlighted and critically assessed.     

Determination of cadmium in grains by isotope dilution ICP–MS and coprecipitation using sample constituents as carrier precipitants

A coprecipitation method using sample constituents as carrier precipitants was developed that can remove molybdenum, which interferes with the determination of cadmium in grain samples via isotope dilution inductively coupled plasma mass spectrometry (ID-ICPMS). Samples were digested with HNO₃, HF, and HClO₄, and then purified 6 M sodium hydroxide solution was added to generate colloidal hydrolysis compounds, mainly magnesium hydroxide. Cadmium can be effectively separated from molybdenum because the cadmium forms hydroxides and adsorbs onto and/or is occluded in the colloid, while the molybdenum does not form hydroxides or adsorb onto the hydrolysis colloid. The colloid was separated by centrifugation and then dissolved with 0.2 M HNO₃ solution to recover the cadmium. The recovery of Cd achieved using the coprecipitation was >97%, and the removal efficiency of Mo was approximately 99.9%. An extremely low procedural blank (below the detection limit of ICPMS) was achieved by purifying the 6 M sodium hydroxide solution via Mg coprecipitation using Mg(NO₃)₂ solution. The proposed method was applied to two certified reference materials (NIST SRM 1567a wheat flour and SRM 1568a rice flour) and CCQM-P64 soybean powder. Good analytical results with small uncertainties were obtained for all samples. This method is simple and reliable for the determination of Cd in grain samples by ID-ICPMS. Figure Overview of a coprecipitation method using sample constituents     

Development of analytical procedures for determination of total chromium by quadrupole ICP–MS and high-resolution ICP–MS,and hexavalent chromium by HPLC–ICP–MS,in different materials used in the automotive industry

A European directive was recently adopted limiting the use of hazardous substances such as Pb, Hg, Cd, and Cr(VI) in vehicle manufacturing. From July 2003 a maximum of 2 g Cr(VI) will be authorised per vehicle in corrosion-preventing coatings of key components. As no standardised procedures are available to check if produced vehicles are in agreement with this directive, the objective of this work was to develop analytical procedures for total chromium and Cr(VI) determination in these materials. The first step of this study was to optimise digestion procedures for total chromium determination in plastic and metallic materials by inductively coupled plasma mass spectrometry (ICP–MS). High resolution (HR) ICP–MS was used to examine the influence of polyatomic interferences on the detection of the ⁵²Cr⁺ and ⁵³Cr⁺ isotopes. If there was strong interference with m/z 52 for plastic materials, it was possible to use quadrupole ICP–MS for m/z 53 if digestions were performed with HNO₃+H₂O₂. This mixture was also necessary for digestion of chromium from metallic materials. Extraction procedures in alkaline medium (NH₄⁺/NH₃ buffer solution at pH 8.9) assisted by sonication were developed for determining Cr(VI) in four different corrosion-preventing coatings by HPLC–ICP–MS. After optimisation and validation with the only solid reference material certified for its Cr(VI) content (BCR 545; welding dusts), the efficiency of this extraction procedure for screw coatings was compared with that described in the EN ISO 3613 standard generally used in routine laboratories. For coatings comprising zinc and aluminium passivated in depth with chromium oxides the extraction procedure developed herein enabled determination of higher Cr(VI) concentrations. This was also observed for the screw covered with a chromium passivant layer on zinc–nickel. For coating comprising a chromium passivant layer on alkaline zinc the standardized extraction procedure was more efficient. In the case of painted metallic plate, because of a reactive matrix towards Cr(VI), its extraction without degradation was difficult to perform.     

Determination of arsenic species and arsenosugars in marine samples by HPLC–ICP–MS

Arsenic-speciation analysis in marine samples was performed by high-pressure liquid chromatography (HPLC) with ICP–MS detection. Separation of eight arsenic species—As^III, MMA, DMA, As^V, AB, TMAO, AC and TeMAs⁺—was achieved on a C₁₈ column with isocratic elution (pH 3.0), under which conditions As^III and MMA co-eluted. The entire separation was accomplished in 15 min. The HPLC–ICP–MS detection limits for the eight arsenic species were in the range 0.03–0.23 μg L⁻¹ based on 3σ for the blank response (n=5). The precision was calculated to be 2.4–8.0% (RSD) for the eight species. The method was successfully applied to several marine samples, e.g. oysters, fish, shrimps, and marine algae. Low-power microwave digestion was employed for extraction of arsenic from seafood products; ultrasonic extraction was employed for the extraction of arsenic from seaweeds. Separation of arsenosugars was achieved on an anion-exchange column. Concentrations of arsenosugars 2, 3, and 4 in marine algae were in the range 0.18–9.59 μg g⁻¹. This paper was presented at the European Winter Conference 2005     

Simultaneous determination of deoxynivalenol,zearalenone, and their major masked metabolites in cereal-based food by LC–MS–MS

Cereals and cereal-based food have often been found to be contaminated with the mycotoxins deoxynivalenol (DON) and zearalenone (ZON), after infection of the grain with the pathogenic fungus Fusarium. Both the pathogen and the infected plants can chemically modify DON and ZON, including acetylation, glucosidation, and sulfation. Analytical strategies for detection and quantification of DON and ZON are well known and established but often fail to recognize the respective metabolites, which are, therefore, also referred to as “masked” mycotoxins. However, several masked forms are also known to be harmful to mammals. Failure to detect these could lead to significant underestimation of the toxic potential of a particular sample. To monitor the levels of DON and ZON metabolites in cereals and cereal-based food, we have developed a LC–MS–MS method capable of simultaneous determination of DON, ZON, and eight of their masked metabolites, namely deoxynivalenol-3-glucoside (D3G), 3-acetyl-deoxynivalenol (3ADON), zearalenone-4-glucoside (Z4G), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), α-zearalenol-4-glucoside (α-ZG), β-zearalenol-4-glucoside (β-ZG), and zearalenone-4-sulfate (Z4S). The suitability of several cleanup strategies including C₁₈-SPE, primary and secondary amines (PSA), MycoSep push-through columns, and immunoaffinity columns was evaluated. The final method used no sample cleanup and was successfully validated for four cereal-based food matrices, namely cornflour, porridge, beer, and pasta, showing good recoveries and precision for all analytes.     

Determination of phosphorus in small amounts of protein samples by ICP–MS

Inductively coupled plasma mass spectrometry (ICP-MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 micro g) or digest (1-10 micro L) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-SFMS) and quadrupole-based ICP-MS (ICP-QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8+/-0.4 mg g(-1), was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP-QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP-MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1-100 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP-SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.The detection limits for phosphorus in biological samples were determined by ICP-SFMS down to the ng g(-1) level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP-SFMS in comparison to ICP-QMS.     

Effects of γ-sterilization on butyltin homogeneity and content in sediments: a GC–ICP–MS study

A GC-ICP-MS method based on extraction and alkylation of butyltins with sodium tetraethylborate was used to quantitatively assess the fate of these analytes in solutions and sediments following exposure to gamma-irradiation. The effects of a 2.5 Mrad sterilization dose on three butyltin species in both methanolic calibration solutions and in sediment matrices were investigated. Although significant losses of tributyltin (TBT, 90%), dibutyltin (DBT, 100%) and monobutyltin (MBT, 80%) were detected in standard solutions prepared in methanol following gamma-irradiation, no species inter-conversion occurred. Some degradation of TBT (38%) and DBT (32%) but no significant change in MBT content was found using a spiked sediment CRM HISS-1. Conversion DBT to MBT in spiked HISS-1 was deduced. Much smaller degradation of TBT (16% loss) and 10% loss of DBT by conversion to MBT (14% gain) was registered using a sediment blend of PACS-2 and HISS-1 (SOPH). Despite some initial losses of TBT and DBT due to irradiation, better than 2% RSD in both TBT and DBT concentrations measured in twelve different bottles of blended sediment SOPH were obtained, indicating the material may be considered homogeneous for these analytes. Results from a long-term five-year stability study of PACS-2 show that all three butyltins are stable during storage at 4 degrees C followed with 2.5 Mrad minimum dose of gamma-irradiation sterilization treatment.     

Simultaneous determination of six major ergot alkaloids and their epimers in cereals and foodstuffs by LC–MS–MS

This paper describes a new and rapid method for accurate quantification of the six ergot alkaloids, ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine, by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The six ergot alkaloids studied have been defined by the European Food Safety Authority (EFSA) as among the most common and physiologically active ones. In addition, the method enables the quantification of the corresponding six epimers (ergo-inines) of these ergot alkaloids. This is of considerable importance in terms of the differences in toxicity of the isomeric forms. The method involves extraction under alkaline conditions using a mixture of acetonitrile and ammonium carbonate buffer followed by a rapid clean-up using dispersive solid-phase extraction with PSA (primary secondary amine) and a short chromatographic LC-run (21 min) with subsequent MS–MS detection. The method was developed and validated using ten different cereal and food samples. The major strength of the new method compared with previously published techniques is the simplicity of the clean-up procedure and the short analysis time. The limits of quantification were 0.17 to 2.78 μg kg⁻¹ depending on the analyte and matrix. Recovery values for the 12 ergot alkaloids spiked into ten different matrices at levels of 5, 50, and 100 μg kg⁻¹ were between 69 and 105% for 85 of 90 recovery measurements made over six days. Measurement uncertainty values were highly satisfactory. At a concentration level of 5 μg kg⁻¹ the expanded measurement uncertainty ranged from ±0.56 to ±1.49 μg kg⁻¹, at a concentration level of 100 μg kg⁻¹ the expanded measurement uncertainty ranged from ±8.9 to ±20 μg kg⁻¹. Both LOQs and measurement uncertainties were dependent on the analyte but almost independent of the matrix. The method performance was satisfactory when tested in a mini-intercomparison study between three laboratories from three different countries. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-throughput analysis of phthalate esters in human serum by direct immersion SPME followed by isotope dilution–fast GC/MS

Solid-phase microextraction (SPME) coupled to gas chromatography/mass spectrometry (GC/MS) was applied to the determination of phthalate esters in human serum. The present method decreased the sample preparation time by a factor of 50 by using direct immersion SPME with an 85-m polyacrylate fiber to extract phthalate esters from the matrix. The use of fast GC/MS further improves total analysis time when compared to other techniques. Isotope dilution was successfully applied to improve the precision, reproducibility, and repeatability of the SPME method. The linear dynamic range spans several orders of magnitude from low ppb to ppm levels, and the LOD for the method is 15 pg L^–1 on average with RSDs less than 4% for the six phthalate esters included in this study.     

Simultaneous determination of six constituents in Mahuang Fuzi Xixin by UPLC-PDA–MS/MS

Mahuang Fuzi Xixin (MFX), a classic recipe in traditional Chinese medicine, belongs to an exterior-relieving formula. For quality control of the MFX products, qualitative analysis using ultra-high performance liquid chromatography with diode-array detector–tandem mass spectrometry (UPLC-PDA–MS/MS) was undertaken. Six compounds from the MFX were simultaneously detected. Among them, astragalin and kaempferol 3-rutinoside were quantified through the UPLC–MS/MS method, while asarinin, sesamin, kakuol and methyleugenol were quantified through the UPLC-PDA method. This method can be applied to the quantitative determination of the six compounds in the MFX.     

Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry

We have developed a simple and sensitive method for the simultaneous determination of testosterone (TES), cortisol (CRT), and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a Discovery HS F5 column. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL/min using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The in-tube SPME LC–MS/MS method showed good linearity with correlation coefficients r?≥?0.9998 for TES, CRT, and DHEA using their respective stable isotope-labeled internal standards. The intra-day and inter-day precisions (relative standard deviations) were below 4.9 and 8.5 % (n?=?5), respectively. This method was successfully utilized to analyze TES, CRT, and DHEA in saliva samples without any other pretreatment or interference peaks, and the quantification limits (S/N?=?10) of TES, CRT and DHEA were about 0.01, 0.03 and 0.29 ng/mL saliva, respectively. The recoveries of these compounds spiked into saliva samples were each above 94 %. This method was applied to analyze changes in salivary TES, CRT, and DHEA levels resulting from stress and fatigue load.     

Analytical development for analysis of pharmaceuticals in water samples by SPE and GC–MS

An analytical procedure involving solid-phase extraction (SPE) and gas chromatography–mass spectrometry (GC–MS) has been developed for determination of pharmaceutical compounds (aspirin, caffeine, carbamazepine, diclofenac, ketoprofen, naproxen, ibuprofen, clofibrate, clofibric acid, and gemfibrozil) in a variety of aqueous samples (wastewater and surface water). After filtration, samples were extracted and concentrated using C₁₈ or HLB cartridges, depending on the type of compound. Sample storage conditions were checked and optimized to ensure preservation of the pharmaceutical substance, taking into consideration environmental sampling conditions. For most of the pharmaceuticals monitored, recovery was in the range 53 to 99% and the variability was below 15% for the complete procedure, with limits of detection ranging from 0.4 to 2.5 ng L⁻¹, depending on the compound. The methods were successfully applied to monitoring of pharmaceutical contamination of the Seine estuary. Concentrations varied from several dozens of nanograms per liter for surface waters to several hundreds of nanograms per liter for wastewaters.     

Simultaneous determination of eight adulterants in weight management supplements and herbs by HPLC–DAD and LC–MS/MS

An efficient HPLC–DAD method was developed for simultaneous determination of eight adulterants in weight management supplements and herbs. The eight adulterants were phenolphthalein, sibutramine, nuciferine, and five anthraquinone compounds including aloe-emodin, rhein, emodin, chrysophanol, and physcion. The analytes were ultrasonically extracted with 70% (v/v) methanol aqueous solution followed by centrifugation. The supernatant was subjected to HPLC analysis. A Phenomenex Luna C18 column was applied for chromatographic separation. The mobile phase was consisted of methanol and aqueous solution of 0.05% (v/v) phosphoric acid–0.025% (m/v) sodium dodecyl sulfonate. The flow rate of mobile phase was 0.8?ml?min^?1 with gradient elution. Clenbuterol and ibuprofen were used as internal standards. The retention times and the characteristic UV spectrograms were used for qualitative analysis. Quantifications were based on the internal standard curves. Good linearities (r?>?0.9996) for all analytes were obtained with the intra- and inter-day precision (n?=?6) ranging from 0.76 to 5.9% and 0.90 to 8.1%, respectively. The average recoveries from the spiked samples with different matrices varied from 73.4 to 114%. Validations were subsequently performed using LC–MS/MS. The proposed method successfully determined the target adulterants in eight commercial weight management supplements and five weight reducing herbs with satisfactory results.     

Analysis of ochratoxin A in grapes,musts and wines by LC–MS/MS: First comparison of stable isotope dilution assay and diastereomeric dilution assay methods

Ochratoxin A (OTA) exhibits potent nephrotoxic, carcinogenic and teratogenic effects and its maximum level in wines has been set to 2 μg L⁻¹ by regulation. Consequently, the analytical procedures for OTA determination in wines have to be both very sensitive and reliable. In this paper, we compared two quantification methods: the stable isotope dilution assay (SIDA) and the diastereomeric dilution assay (DIDA). For this purpose, non-natural analogues of OTA were synthesized: the labeled OTA (OTA-d₄) as a diastereomeric mixture for the SIDA and one non-natural OTA’s diastereomer (OTA-dia) for the DIDA. To quantify OTA in red grapes, musts or wines, the sample preparation was optimized using immunoaffinity column extraction and the analysis was performed by LC–MS/MS in Multiple Reaction Monitoring mode. A validation procedure in agreement with the International Organization of Vine and Wine recommendations was conducted. It appeared that SIDA quantification exhibited excellent sensitivity (LOD < 1 ng L⁻¹), accuracy (recovery = 98%), repeatability (RSD < 3%) and intermediate reproducibility (RSD < 4%) compared to quantification by DIDA. Indeed, DIDA method did not provide satisfactory results demonstrating that immunoaffinity extraction is exclusively selective for the natural OTA and not for its diastereomer, which therefore cannot be considered as a good internal standard for this particular method.