Isotope-labeled internal standards

It is possible to carry out a chromatographic separation, collect all, or selected, fractions and then, after removal of the majority of the volatile solvent, transfer the analyte to the mass spectrometer by using the conventional inlet (probe) for solid analytes. The direct coupling of the two techniques is advantageous in many respects, including the speed of analysis, the convenience, particularly for the analysis of multi-component mixtures, the reduced possibility of sample loss, the ability to carry out accurate quantitation using isotopically labelled internal standards, and the ability to carry out certain tasks, such as the evaluation of peak purity, which would not otherwise be possible. 

There are two notable features of the quantitative performance of this type of interface. It has been found that non-linear responses are often obtained at low analyte concentrations. This has been attributed to the formation of smaller particles than at higher concentrations and their more easy removal by the jet separator. Signal enhancement has been observed due to the presence of (a) coeluting compounds (including any isotopically labelled internal standard that may be used), and (b) mobile-phase additives such as ammonium acetate. It has been suggested that ion-molecule aggregates are formed and these cause larger particles to be produced in the desolvation chamber. Such particles are transferred to the mass spectrometer more efficiently. It was found, however, that the particle size distribution after addition of ammonium acetate, when enhancement was observed, was little different to that in the absence of ammonium acetate when no enhancement was observed. 

Wheat samples are extracted with dilute ammonia on the ASE200. The extracts are amended with isotopically labeled internal standards. The extracts are purified by sequential octadecyl reversed-phase solid-phase extraction (Cig SPE) and ethylenediamine-iV-propyl anion exchange (PSA) SPE. The samples are analyzed by LC/MS/MS. This method determines crop residues of flucarbazone-sodium and A-desmethyl flucarbazone with a limit of quantitation (LOQ) of 0.01 mgkg for each analyte. 

The wheat sample residue level is determined from the relative mass spectral responses of the analytes to the corresponding isotopically labeled internal standards. The sample relative response is compared with the average relative response of a standard solution of analyte and internal standard analyzed before and after the sample (bracketing standards). Both samples and standards receive the same amount, 100 ng, of each internal standard to facilitate the comparison. The calculations to determine the residue level in wheat tissues are outlined in Section 7.3.1. 

Residues of isoxaflutole, RPA 202248 and RPA 203328 are extracted from surface water or groundwater on to an RP-102 resin solid-phase extraction (SPE) cartridge, then eluted with an acetonitrile-methanol solvent mixture. Residues are determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Cg column. Quantitation of results is based on a comparison of the ratio of analyte response to isotopically labeled internal standard response versus analyte response to internal standard response for calibration standards. 

Macerated plant material is homogenized with acetone-water (3 1, v/v) and vacuum Altered, and the Alttate is adjusted to constant volume. A portion of the Altrate is further Altered through a syringe Alter and diluted 1 1 with an isotopically labeled internal standard solution for analysis by electrospray LC/MS/MS. 

Soil samples are extracted with methanol-water (7 3, v/v) using a Soxtec extractor. After addition of an isotopically labeled internal standard (IS) and dilution to 50 mL, the extracts are analyzed by electrospray LC/MS/MS. 

Whole blood Purge-and-trap pre-concentration (with isotopically-labelled internal standard) GC/lsotope dilution MS 0.040 ppb 105 at 0.054 ppb Ashley et al. 1992... 

Liang, H.R. Foltz, R.L. Meng, M. Bennett, P. Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 2815—2821. 

Following extractive deproteinization of the plasma, the amino acids (and their stable-isotope-labeled internal standards) are separated by HPLC and introduced into the mass spectrometer. Electrospray ionization results in the formation of electrically charged molecules, which are separated on the basis of their mass/charge (m/z) ratio in the first quadrupole. Following fragmentation in the collision cell, the characteristic fragment for each amino acid is selected in the second quadrupole. This process is named multiple reaction monitoring. 

Table 2.1.3 Multiple reaction monitoring of amino acids for their tandem mass spectrometry quantitation. In daily practise not all mentioned amino acids are measured in one run, but a set of ten dedicated evaluation programs has been developed, covering groups of amino acids associated with groups of disorders. Amino acids presented in italics indicate stable-isotope-labeled internal standards ... 

Reference values of this approach are not different from those for other amino acid analyses. An example of a mass chromatogram, representing the plasma of a PKU patient, is shown in Fig. 2.1.1. When evaluating the results of MS/MS amino acid analyses, one has to reahze that the hquid chromatographic separation is by far less efficient that the AAA separation. For this reason, any amino acid may (partly) coelute with other amino acid(s), which potentially interferes with its mass spectromet-ric behavior. This effect is known as quenching. In order to overcome this as much as possible, stable-isotope-labeled internal standards (as many as possible) should be used. However, this matrix effect of ion suppression is the major pitfall in the MS/MS analysis of amino acids. Consequently, the MS/MS analysis of amino acids cannot be regarded as a reference method, similar to all other amino acid analytical methods. 

Fig. 2.1.1 Tandem mass spectrometry analyses of plasma phenylalanine (phe) and tyrosine (tyr) in a patient with phenylketonuria (PKU left panel phe 793 pmol/1, tyr 70 pmol/1) and in a control (right panel phe 27 pmol/1, tyr 28 pmol/1). The stable-isotope-labelled internal standards are D4-tyrosine (D4-tyr), containing four deuterium atoms and D5-phenylalanine (D5-phe), which has five deuterium atoms.

SIM average detection limit (with stable-isotope-labeled internal standard 0.01 mmol/mol creatinine. Reproduced from reference [32], with permission. nd Not detected... 

Table 3.1.10 (continued) Reference ranges of organic acids and acylglycines in urine in children of different ages. Values are presented as mmol/mol creatinine. TIC average detection limit 0.1 mmol/mol creatinine SIM average detection limit (with stable-isotope-labeled internal standard 0.01 mmol/mol creatinine. Reproduced from reference [32], with permission. nd Not detected... 

Measured using a stable-isotope-labeled internal standard... 

Acylcarnitine analysis using stable-isotope-labeled internal standards provides quantitative data for acylcarnitine species [14]. However, to provide meaningful results to referring healthcare providers, it is critical to complement analytical proficiency with in-depth interpretation of results, as is true for many other examples of complex metabolic profiles [39]. 

Additional or different labeled and unlabeled standards can be used. However, note that the incubation as described here includes isotopically labeled L-valine and L-isoleucine and that the metabolites of these branched chain amino acids can interfere with isotopically labeled internal standards for butyrylcarnitine or propi-onylcarnitine. 

There are a finite number of analytes that have been measured commercially for many years for the diagnosis of steroid synthetic and metabolic disorders. The discussion of analytes and methodologies below is restricted to these analytes, as listed in Table 5.3.1. This table summarizes the best quantitative normative values available at the time of writing. Accurate values of most steroids listed have been obtained by MS/MS quantitation using isotope-labeled internal standards. 

Guo and co-workers [24,25] have spearheaded the development of MS/MS serum steroid profiles. Their most recent report describes profiling in 11 min of 12 steroids in 200 pi serum with minimal work-up, comprising acetonitrile protein precipitation. The steroids analyzed were as follows DHEA sulfate, DHEA, aldosterone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, estradiol, testosterone, 17-hy-droxyprogesterone, progesterone, and 25-hydroxyvitamin D3. Stable-isotope-labeled internal standards were incorporated for each steroid. An API-5000 instrument was used with the APPI source in positive-ion mode, with the exception of aldosterone, which had greater sensitivity in negative-ion mode. Separation was carried out on a C8 column, which allowed more rapid separation than the more commonly utilized C18. The MRM transitions utilized are shown in Table 5.3.1. The lower level of sensitivity was between 1.5 and 10 pg/ml, dependent on the steroid. The authors were exhaustive in addressing issues of accuracy, recovery (90-110%) and reproducibility (< 12.2% for same-day and between-day). 

Palermo M, Gomez-Sanchez C, Roitman E, Shackleton CH (1996) Quantitation of cortisol and related - -4-ene steroids in urine using gas chromatography/mass spectrometry with stable isotope-labeled internal standards. Steroids 61 583-589... 

Perhaps no reagent is more important than the stable-isotope-labeled internal standard in any clinical assay utilizing mass spectrometry for quantification. Internal standards are important in many aspects of the analysis and are somewhat different than standards utilized in other clinical, non-mass-spectrometric assays. The ideal internal standard is an enriched isotopic version of the analyte being measure. For example, in the case of phenylalanine, a standard available may contain six 13C molecules rather than 12C in the aromatic ring. This has the net effect of shifting the mass of phenylalanine by six units while also maintaining nearly identical chemical... 

Stokvis, E., Rosing, H., and Beijnen, J. H. (2005). Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry Necessity or not Rapid Commun. Mass Spectrom. 19 401-407. 

Lanckmans, K., Sane, S., Smolders, I., and Michotte, Y. (2007). Use of a structural analogue versus a stable isotope labeled internal standard for the quantification of angiotensin IV in rat brain dialysates using nano-liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 21 1187-1195. 

Wang, S., Cyronak, M., and Yang, E. (2007). Does a stable isotopically labeled internal standard always correct analyte response A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma. J. Pharm. Biomed. Anal. 43 701-707. 

Soil, sediment (CDDs) Addition of isotopically labeled internal standards to sample addition of Na2S04 and extraction with hexane/methanol or Soxhlet extraction with toluene clean-up using column chromatography if needed volume reduction HRGC/MS (EI/SIM) No data No data Eschenroeder et al. 1986... 

Abstract Internal standards play critical roles in ensuring the accuracy of reported concentrations in LC-MS bioanalysis. How do you find an appropriate internal standard so that analyte losses and experimental variations during sample preparation, chromatographic separation, and mass spectrometric detection could be corrected How is the concentration of an internal standard determined Should internal standard responses be monitored during the analysis of incurred samples What are the main causes for internal standard response variations How do they impact the quantitation Why are stable isotope labeled internal standards preferred And yet one should still have an open-mind in their usage for the analysis of incurred samples. All these questions are addressed in this chapter supported by theoretical considerations and practical examples. 

There are two main types of internal standards. The first ones are stable isotope labeled (SIL) internal standards. They are compounds in which several atoms in the analytes are replaced by their respective stable isotopes, such as deuterium (2H, D or d), 13C, 15N, or 170. Labeling with the first three isotopes are most common, particularly labeling with deuterium (due to less difficulty in synthesis and therefore less expensive). For examples, raloxifene-d4-6-glucuronide was used as the internal standard for the determination of raloxifene-6-glucuronide [5] and 1, 2, 3, 4-13C4 estrone (PCJEl) was used as the internal standard for estrone (El) [6], The usage of stable isotope labeled internal standards in quantitative LC-MS or GC-MS analysis is often termed as isotope dilution mass spectrometry (IDMS) [7],... 

Stable Isotope Labeled Internal Standards vs. Structural Analogue Internal Standards... 

Internal standards play critical roles in ensuring the accuracy of final reported concentrations in quantitative LC-MS bioanalysis through the correction of variations during sample preparation, LC-separation, and MS detection. The physical-chemical properties of an internal standard, particularly hydrophobicity and ionization properties should be as close as possible to those of the corresponding analyte to better track the variations the analyte experiences. For this reason, stable isotope labeled internal standards should be used whenever possible. However, efforts should still be made to obtain clean extracts, adequate chromatographic separation, and optimized ionization mode and conditions. 

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. 

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