Analogue internal standards

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

As demonstrated by many studies, SIL internal standards outperform structural analogue internal standards in terms of precision and accuracy provided the extraction and LC separation are comparable [8, 25-30], In addition, SIL internal standards can also extend linearity range, enable the usage of less reliable transitions (e.g., loss of water), and prolong in-processing and postpreparation stabilities [8, 28],... 

A similar method using a structure analogue internal standard (D-24843, which is the identical molecules as D-24851 but lacking the chlorine atom), has been developed as well. However, the overall performance of the assay in terms of accuracy and precision was clearly inferior compared to the presented method using the labeled internal standard. 

While the selection of an isotopically-labelled or analogue internal standard is relatively easy in quantitative bioanalysis, the situation is more complicated in multiresidue analysis. It is difficult to select appropriate analogue standards for a wide variety of target compounds, while isotopically-labelled standards are often not available for all target compounds. In addition, if one would introduce one standard per target compound, this would seriously limit the sensitivity of the method as it doubles the number of SRM transitions that have to be monitored. Another problem in multiresidue analysis is the selection of appropriate blanks for the production of the matrix-matched standards and the number of matrices that might have to be studied. When no adequate blank matrix is available, the standard addition method is the only way to achieve sufficiently accurate and precise results [123]. This method is time-consuming and labourious. 

In order to obtain sufficient accurate results with good precision, an internal standard (IS) must be used in quantitative bioanalysis using LC-MS. Either an isotopically-labelled internal standard (ILIS) or an analogue internal standard (ANIS) can be apphed. 

The advantage of using a mass spectrometer as the detector is associated with cases (ii) and (iii) above. In particular, because mass may be used as a discriminating feature, it is possible to use an isotopically labelled analyte as an internal standard. These have virtnally identical physical and chemical properties to the unlabelled analogue, and are therefore likely to experience similar losses during... 

Adequate precision and accuracy are only likely to be achieved if some standardization procedure is employed and the nature of this, internal or external standards or the method of standard additions, needs to be chosen carefully. If internal standardization procedures are adopted then appropriate compound(s) must be chosen and their effect on the chromatographic and mass spectrometry methods assessed. The ideal internal standard is an isotopically labelled analogue of the analyte but, although there are a number of commercial companies who produce a range of such molecules, these are not always readily available. An analytical laboratory is then faced with the choice of carrying out the synthesis of the internal standard themselves or choosing a less appropriate alternative with implications on the accuracy and precision of the method to be developed. 

In the absence of any added salts, the APCI-MS spectra were dominated by the Na+ adducts, as shown in Fig. 2.8.5. The NH4 and K+ adducts were present at lower intensities, the latter especially for the higher molecular weight analogues. Addition of CH3CO2NH4 did not simplify the adduct formation to [M + NH4]+ species as observed in ESI-MS and the best results for APCI-MS analysis were obtained without addition of any salt solutions. Application of this method to determinations of M2D-C3-0-(E0)n-Me recovery from solid substrates was achieved, using triethylene glycol monohexyl ether [C6(EO)3] as the internal standard (Fig. 2.8.5) [29],... 

Internal standards are used in quantitative work in a similar manner to GLC. These are usually homologues or isotopically labelled analogues of the analyte, e.g. deuterium (2H). 

Although DIOS-MS is mainly a tool for qualitative analysis, many examples have shown that quantitative analysis is possible when internal standards are used. These may either be isotope-labelled - mostly deuterated - compounds or structurally related analogues. For example, subsequent to electrospray deposition, amino acids such as phenylalanine and tyrosine have been successfully quantified by means of DIOS-MS using their deuterated analogues as internal standards. 

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... 

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. 

The second ones are structural analogues with different masses or even the same mass. In the latter case, chromatographic separation between the analyte and its internal standard must be achieved when distinctive MRM (multiple reaction monitoring) transitions could not be found. It is preferable that the key structure and functionalities (e.g., -COOH, -S02, -NH2, halogens, and heteroatoms) of an internal standard are the same as those of the analyte and differ only by C-H moieties (length and/or position). Modifications in functionalities would result in significant differences in ionization efficiency and extraction recovery [8],... 

Despite the very desirable performances of SIL internal standards, they are not always available or are very expensive, especially when seeking exclusively non-deuterium-labeled internal standards. Then, structural analogues can be used. In this case, coelution of an analyte and its internal standard is preferred in order to reduce matrix effect [11] or to expand linearity range [12],... 

Once potential structural analogues are found, their physical-chemical properties, such as log/) (hydrophobicity) vs. pH, can be calculated and compared with those of the analyte using software (e.g., Pallas) prior to being experimentally tested. As shown in Fig. 2, both acyclovir and ganciclovir could be used as the internal standard for penciclovir, particularly the latter due to the same number of hydroxy... 

Unfortunately, for the majority of small molecule LC-MS/MS analyses, stable isotope labelled internal standards are not available so far. In such cases, compounds with a very similar molecular structure typically serve as internal standard ( homologues or analogues ). Since the ionization properties are substantially determined by functional groups of a molecule, ionization behaviour may differ significantly—even between compounds with very similar over-all molecular structure. Differential clustering, e.g. with sodium, ammonium or formate ions often present in mobile phases may as well impact the parity of ionization yield between analyte and internal standard. Hence the availability of an appropriate homologue is crucial and critical for the development of reliable LC-MS/MS methods in TDM [51]. 

Another requirement for qualitative or quantitative analysis is the use of internal standards (IS) to compensate for sample preparation or chromatographic variability. This is of particular importance in LC-MS analysis, as an adequate IS can also compensate for the negative influence of matrix effects on method precision and accuracy. Stable-isotope-labeled ISs are the most appropriate for this purpose. If a specific deuterated analogue is not commercially available, it could be substituted for deuter-ated substances with similar physicochemical properties to the analyte of interest. However, the use of other marketed pharmaceuticals for this purpose should be avoided, as it cannot be excluded that the patient to be monitored has taken that drug. 

The internal standard should show physical and chemical properties that are as close as possible to those of the molecule that has to be measured. It must be pure, absent from the sample and, of course, inert towards the compounds in the sample. The internal standards can be classified into three categories structural analogues that are labelled with stable isotopes, structural homologues and compounds from the same chemical family. These various types of internal standards are classified here in descending order according to their usefulness and their price. In fact, the starting material for labelled compounds is fairly... 

A further advantage of combining IPC and MS is that isotope-labeled internal standards may be used for accurate quantification of analytes. No recovery check is needed for analytes because the labeled analogues of the analytes that serve as internal standards are added to the samples before the extraction step. This allows sample mixtures to be analyzed without pretreatment because the labeled internal standards share the fate of the un labeled analyte during sample processing without differences in extraction efficiency between the labeled standards and unlabeled analytes. This method achieved excellent precision and improved linearity of calibration lines despite interference from sample matrices in the quantitative analysis of important secondary intracellular metabolites in a complex biological sample solution from cultured cells. The technique is a valuable strategy for metabolomics [75],... 

If a mass spectrometer is being used as the detector, then the ideal internal standard is a analogue of the drug. 

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