Standards, internal deuterated

Acetochlor, alachlor, and metolachlor are determined in ground and surface water samples. Deuterated internal standards are added to each water sample, and analytes are extracted using an SPE column. After elution and concentration to an appropriate volume, the analytes are quantitated by GC/MS. 

Analytical accuracy. The mixture of all deuterium-labeled internal standards is added to each water sample before extraction. This does not prevent the loss of the unlabeled herbicides from the sample in subsequent processing steps, but a proportional loss of the deuterated internal standard precludes the need to correct for recovery. Although referring to recovery in this type of analysis is inappropriate, the accuracy of this method should be monitored. 

Combine a 0.5-mL aliquot of the Anal sample exAact or a 0.020 igmL azinphos-methyl standard solution in acetone-water (2 1 v/v) with 0.5 mL of a 0.040 ig mL deuterated internal standard solution in methanol-water (2 3 v/v) in an HPLC autosampler vial. Combination may be made using other volumes as long as the solutions are combined 1 1 (v/v). Inject 200 aL from the 0.020 and 0.040 igmL standard/internal standard soluAon. Inject 200 aL from each of the 10 sample exAact/ internal standard soluAons. Inject 200 p-L from anoAier 0.020 and 0.040 pgmL standard/internal standard solution. 

Greaves et al. [74] used a selected ion-monitoring assay method for the determination of primaquine in plasma and urine using gas chromatography-mass spectrometric method and a deuterated internal standard. After freeze-drying and extraction with trichloroethylene, the sample plus internal standard was reacted with Tri Sil TBT (a 3 3 2 by volume mixture of trimethylsilylimidazole, A/O-bis-(trimethylsilylacetamide and trimethylchlorosilane) and an aliquot injected to the gas chromatograph-mass spectrometer. The gas chromatographic effluent was monitored at m/z 403, and m/z 406, the molecular ions of the bis-tetramethylsilane ethers of primaquine and 6-trideuteromethoxy primaquine. 

Quantification is usually achieved by a standard addition method, use of labeled internal standards, and/or external calibration curves. In order to allow for matrix interferences the most reliable method for a correct quantitation of the analytes is the isotope dilution method, which takes into account intrinsic matrix responses, using a deuterated internal standard or carbon-13-labeled internal standard with the same chemistry as the pesticide being analyzed (i.e., d-5 atrazine for atrazine analysis). Quality analytical parameters are usually achieved by participation in interlaboratory exercises and/or the analysis of certified reference materials [21]. 

Lopez-Avila et al. [59] used microwave assisted extraction to assist the extraction of polyaromatic hydrocarbons from soils. Another extraction method was described by Hartmann [60] for the recovery of polyaromatic hydrocarbons in forest soils. The method included saponification of samples in an ultrasonic bath, partitioning of polyaromatic hydrocarbons into hexane, extract cleanup by using solid-phase extraction, and gas chromatography-mass spectrometric analysis using deuterated internal standards. Polyaromatic hydrocarbons were thermally desorbed from soils and sediments without pretreatment in another investigation [61]. 

A rapid technique for the identification of surfactants in consumer products by ESI-MS was proposed by Ogura and co-workers [6], After a simple preparation procedure, infusion of the sample, which was prepared in a water/methanol mixture (50 50) containing 10 mM ammonium acetate, allowed assignment of the [M + NH4]+ ions of Cio- and Ci2-mono- and -diglucoside in the mass spectrum (ion masses as in Table 2.7.1). The approach even permitted quantitative analysis when deuterated internal standards were used. 

The development of an easy-to-handle method for the qualitative and quantitative determination of surfactants in consumer products was the goal for applying ESI in the FIA-MS(+/—) mode by direct infusion into the mass spectrometer. In this way Ci2, Ci4, Ci6 and Ci8 ASs could be determined besides other anionics (LASs, alkylcarboxylates), nonionics (alkyl polyglucosides (APGs)) and cationics (quats and ester-quats). The methods applied for concentration and determination (MS-MS) helped to identify the compounds and in addition deuterated internal standards were applied for confirmation [57]. 

Jacobson, GA., Chong, E.V., and Davies, N.W., LC-MS method for the determination of albuterol enantiomers in human plasma using manual solid-phase extraction and a non-deuterated internal standard, J. Pharm. Biomed. Anal., 31, 1237, 2003. 

Bulk oils Purge-and-trap preconcentration (with deuterated internal standards) onto Tenax thermal desorption GC/MS 1 ppb (w/v) 88 (6.7% RSD) at 93 ppb Thompson 1994... 

We end our analysis of describing procedures (in submove 2) by examining ways in which authors describe QA/QC procedures in their Methods sections. In general, there are two basic approaches. The first approach embeds the QA/QC procedures in the procedure itself. For example, in excerpt 3P, the authors describe how they added a deuterated surrogate (recovery) standard to their samples at the start of their procedure and how they added a deuterated internal standard at the end of their procedure. The authors go on to describe the results of these procedures in their Results section. 

Air, gloves (surrogate for dermal exposure) Preconcentration from air sample using polyurethane foam (PUF). Soxhlet extraction of PUF or gloves with 5% ethyl ether/hexane. Addition of deuterated internal standards and concentration using K-D and nitrogen blowdown. Capillary GC/MS (can use multiple ion detection) 55 ng/m3 (5.5 nr sample) 73 (14% RSD) Hsu et al. 1988... 

The method measures cortisone (urinary free cortisone, UFE), cortisol (urinary free cortisol, UFF), 6/1-hydroxycortisol, and 18-OHF using deuterated internal standards [62]. Commercial tetradeuterocortisol was used as an internal standard for cortisol, and the remaining dideutero homologs prepared in the laboratory by deuteration of A1 analogues. UFF and UFE are considered better indicators of hormone availability and hypersecretion than the F and E (free plus conjugated) quantified in the comprehensive profile. Typically the values of total F and E are about three times that of... 

Shackleton CHL, Kletke C, Wudy S, Pratt JH (1990) Dehydroepiandrosterone sulfate quantification in serum using high-performance liquid chromatography/mass spectrometry and a deuterated internal standard a technique suitable for routine use or as a reference method. Steroids 55 472-478... 

Table II. Summary of MAS Recovery Data (Including Deuterated Internal Standards)...

Unexpected Results Using Deuterated Internal Standards... 

Despite the overall good performance of SIL internal standards, one must not take it for granted due to the complexity of biological samples, particularly when deuterated internal standards are used. Deuteration could cause differences in hydrophobicity, reaction rates, and noncovalent interactions [31, 32], It is usually observed that a deuterated internal standard elutes slightly earlier than the analyte does in reversed-phase LC. This is even more pronounced with extensive deuteration and long retention time. Sometimes, base-line resolution between an analyte and its deuterated internal standard could be achieved. For example, when D[0 internal standards were used and the retention time was longer than 15 min, pibutidine metabolites were completed separated from their deuterated internal standards (Fig. 5 [33]). 

Sometimes, even the slight separation between an analyte and its deuterated internal standard could cause significant quantitation errors due to differential ion suppression towards the analyte and its deuterated internal standard [34, 35], As shown in Fig. 6, the elution of carvedilol and its internal standard (D5-carvedilol) overlaps with the declining edge of a matrix suppression region in a matrix lot. This resulted in more pronounced ion suppression for the slightly later eluted... 

Fig. 6 Profile of postcolumn infusion of carvedilol with an injection of control plasma extract (lot 3, the problematic lot) overlaid with the LC-MS/MS chromatograms of carvedilol and its deuterated internal standard (D5-carvediol) to demonstrate a significant difference in ion suppression (-25 %) due to even a very small difference in retention time (0.02 min) between carvediolol-S (1.93 min) and its deuterated internal standard (1.91 min). Reproduced from ref. [35] with permission from Elsevier...

Moreover, significant difference in extraction recovery has been observed for an analyte and its deuterated internal standard. For example, the extraction recoveries of haloperidol and d4-haloperidol were 72 % and 44 %, respectively [19], The large difference in extraction recovery could be due to differences in the aforementioned physicochemical properties, such as pA a, or hydrogen-deuterium exchange. 

Since no similar issue has been reported for other SIL internal standards, such as 13C and 15N labeled internal standards, it is therefore necessary to distinguish deuterated internal standards from other SIL internal standards. Nevertheless, one should always have an open mind while using SIL internal standards in LC-MS bioanalysis. 

Fig. 14 (a, top) Hydrophobicity (log D) vs. pH curves for penciclovir and vidarabine. (b, middle) Less internal standard response variation was observed while using vidarabine as the internal standard (CV= 13.02 %) but 43 % of the calibration standards (CS) and quality controls (QC) were rejected. Extraction MCX (mixed-mode strong cation exchange)-based solid-phase extraction, (c, bottom) More IS response variation was observed while using a deuterated internal standard, penciclovir-d4 (CV = 23.81 %) but 100 % of the CS and QC samples were accepted. Reproduced from ref. [36] with permission from Elsevier... 

Stable isotope labeled internal standards may be the best, but they cannot always follow an analyte to compensate the variations of experimental conditions, particularly deuterated internal standards. In addition, low variation in internal standard responses may not be interpreted as good results, though it is favored. Stable internal standard response is good only when it is sure that the internal standard behaves the same way as the analyte does. 

Savard C, Pelletier N, Boudreau N, Lachance S, Levesque A, Masse R (2010) Relative instability of deuterated internal standard under different pH conditions and according to deuterium atoms location. Presented at 58th ASMS conference on mass spectrometry and allied topics, Salt Lake City, Utah, USA, 23-27 May... 

Furthermore, if high ionization temperatures are employed (particularly in APCI), hydrogen-deuterium exchange of deuterated internal standard compounds may occur during the ionization process [50], For that reason, but also due to its typical location in the molecular backbone of an analyte (minimizing the influence on the electronegativity distribution of the scaffold), 13C atoms are considered the more reliable label for isotope dilution internal standardization compared to deuterium. 

Table 2 MRM transitions, cone voltages (CV), and collision energies (CE) used in the analysis of the antidepressants and the deuterated internal standards included in the LC-MS/MS method...

Following the addition of 300 ng of deuterated internal standard (5c, cf. Fig. 2) to 3.0 ml of cold plasma or urine, the sample was sonicated with 30 ml... 

The limit of detection using this methodology is approximately 10 ng. Quantitation is not easily or reliably obtained by this technique. However, the reader is referred to a paper by Clay et al. (1984), in which a deuterated internal standard is used for estimation of PAF. There are certain problems with this approach, unfortunately, and these are addressed in a review by Hanahan and Weintraub (1985). Particular attention is focused on the interaction of the sample with the support matrix, namely, thioglycerol. 

Anticancer Drug) in Human Plasma and Urine by Liquid-Liquid Extraction and High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry, Using a Deuterated Internal Standard. . 613... 

---