Homologue internal standards

A fundamental requirement for LC-MS/MS calibration materials is that matrix effects exerted by these materials are most similar to the matrix effects exerted by actual patients sample materials. Lyophilisation, virus inactivation and other procedures applied during the industrial production of calibration and control materials, may notably impact the ionization behaviour of extracts from such samples and can result in differential matrix effects in calibrators and actual patients samples. If the internal standard peak areas found for calibration samples systematically differ from those found in patients samples, inappropriateness of the calibration materials should be suspected. However, we have previously observed that calibration materials from different commercial sources lead to inaccurate tacrolimus results in an instrument specific manner, without showing deviations in the internal standard peak area. This effect was most likely related to ionization enhancement affecting the target analyte but not the homologue internal standard (ascomycin) ionization and being restricted to calibrator samples. This resulted in systematically low tacrolimus results of clinical samples in one instrument for one specific calibrator lot [52],... 

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). 

Internal standardization circumvents the effects of time-variant instrument response, but does not compensate for different ionization efficiencies of analyte and standard. For internal standardization, a compound exhibiting close similarity in terms of ionization efficiency and retention time is added to the sample at a known level of concentration, e.g., an isomer eluting closely to the analyte or a homologue may serve for that purpose. It is important to add the standard before any clean-up procedure in order not to alter the concentration of the analyte without affecting that of the standard. For reliable results, the relative concentration of analyte and standard should not differ by more than a factor of about ten. 

Internal standards of rather close relationship to the compounds analysed should be used, e.g. homologues (M-i-14) or isotopomers of analytes ( H or labelling), owing to optimal identity of physical or chemical properties (e.g. Ko-vats index in GC). 

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]. 

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... 

All of the specified ions listed in Table 5 for each PCDD/PCDF homologue and labeled standards must be present in the SICP. The ion current response for the two quantitation ions and the M-[COCl]+ ions for the analytes must maximize simultaneously ( 2 seconds). This requirement also applies to the internal standards and recovery standards. For the cleanup standard, only one ion is monitored. 

Catecholamines. The quantitative determination of dopamine and noradrenaline in tissue samples of 0.1-10 mg at levels in the order of 0.5 pmol has been described [84]. These methods are based on extraction, formation of the pentafluorpropionyl derivatives, and the use of the homologues, a-methyidopamine and a-methylnoradrenaline as internal standards in SIM. Higher sensitivity than obtainable with fluorimetric or enzymic assays is reported [462J. Applications have been to amine determination in specific regions of rat brain [84] and to measurement of heart ventricle concentrations [463]. A combination of assays of this type with the use of synthesis inhibitors or radioisotope labelled precursors allows direct estimation of brain amine turnover in animals. 

The internal standard s coefficient of response for the detector used must be of the same order of magnitude as the one of the product to be determined in no way can it be present as an impurity in the sample it must be added at a concentration level that gives a peak area more or less equivalent to the one of the product to be determined. Homologues of the product to be analyzed may be used as internal standards. 

A microcolumn packed with 3 g of activated silica gel is used for sample cleanup and fractionation. An aliquot of the extract (containing about 20 mg oil or TSEM) is then transferred to the silica gel cleanup column to remove polar components and other interferences. The column is eluted first with hexane, which recovers samrated hydrocarbons as Fraction 1 (FI). The mixture of hexane/DCM or hexane/benzene (1 1, v v) is used to elute the aromatic compounds as Fraction 2 (F2). Half of FI and half of F2 are combined into the Fraction 3 (F3). These three fractions are concentrated to appropriate volume (0.5 to 1.0 ml) by nitrogen purge. The quantitation internal standards are 5-a-androstane, di4-terphyl, and C30 17(3(H), 21(3(H)-hopane. The Fraction 3 is analyzed for quantitation of the TPH and the UCM by GC-FID. The Fraction 1 is analyzed for determination of n-alkanes by GC-FID and biomarker terpanes and steranes by GC-MS. The Fraction 2 is analyzed for the determination of alkylated PAH homologues and other EPA priority unsubstantiated PAHs by GC-MS. 

The GC-MS analysis is conducted by injection of 1 /xL of FI orF2 into a gas chromatograph-mass spectrometer. The MS detector is operated in the scan mode to obtain spectral data for identification of components and in the selected ion mode (SIM) for quantitation of target compounds. An appropriate temperature program is selected to achieve near-baseline separation of all of the target components. Quantitation of the alkalized PAH homologues, other EPA priority PAHs and biomarker compounds are performed by the internal standard method with the RRFs for each compound determined during the instrument calibration. The ions monitored for alkylated PAH and biomarker analyses are listed in Table 27.4 and Table 27.5, respectively. 

Elliptinium, the 2-methyl derivative of 9-hydroxyellipticine (Figure 6.11) has been measured in plasma using an ODS-modified silica column and methanol-water (60-1-40) containing ammonium acetate (100 mmol L final pH 6.0) as eluent. Sample preparation was by ion-pair extraction into ethyl acetate containing sodium tetraphenylborate. The 2-propyl homologue was used as the internal standard. Detection was at a GCE (0.6 V V5 Ag/AgCl). The LoD was 250 pg (S/N = 2), equivalent to 25 pgL for a 200 pL sample. 

Internal and External Standards. Quantitative determinations require standards, either external or internal. External standardization using samples with known amounts of analyte ( standard addition ) is possible, if a reproducibility and accuracy of ca. 10% is acceptable. In pharmaceutical studies or routine environmental work such an accuracy is sufficient, since individual variations are much larger. When the required precision must be higher, internal standards must be used. As internal standard a compound with similar chromatographic and. if possible, similar mass spec-trometric properties as the analyte can be employed. This may be a homologue, an analogue with, e.g., a different heteroatom, or a positional isomer. The best choice is an isotopically labeled compound containing stable isotopes. 

The recent dramatic development of mass spectrometry has developed gas chromatography-mass spectrometry (GC-MS) and LC-MS methods. Although pantothenic acid is not volatile enough for direct GC, pantothenic acid can be detected by MS after conversion to volatile compounds such as trimethylsilyl derivatives. Derivatization to volatile compounds from pantothenic acid requires an internal standard and the pantothenic acid homologue hopantothenic acid or radioisotope labelled [ C3, N]-pantothenic acid are used as internal standards for measurement by GC-MS (Banno et al. 1990 Rychlik 2000). 

FAB or LSIMS using a probe inlet does not readily lend itself to quantitative work. Firstly, it is not possible to know how much of the sample has been consumed in the analysis. Secondly, discrimination effects (see section 12.3.3) prevent the comparison of intensities between species of differing surface activity. Semiquantitative results may be readily obtained if discrimination effects are assumed to be constant for the species of interest, for example the determination of homologue distributions in a mixture. For accurate quantitation an internal standard of an isotopically enriched analogue of the analyte should be used. For example, in the determination of cationic surfactants in environmental samples [10], quantitation was achieved by using an internal standard of a trideuterated form of the analyte. In this way the standard will be subject to the same level of discrimination as the analyte. Discrimination effects between different cationic species may also be reduced by adding to the sample an excess of a highly surface-active anionic surfactant. The anionic species will dominate the matrix surface and attract cations into the surface monolayer [10]. 

There are many instances in which quantitative measurements are required in natural product chemistry, particularly in the biomedical field. MS is a well-established tool in this area and offers a unique combination of sensitivity, specificity and dynamic range. In the past GC/MS has usually been the method of choice and procedures and problems involved in such analyses are well documented 1,2, 50). Frequently, the most difficult item in the whole procedure is the choice, and possible synthesis of an appropriate internal standard (homologue, isomer, closely related analogue, or isotopically labelled analogue). Furthermore, the selection of a derivative with suitable GC and MS properties for both sample and standard is required. 

Homologue specific method S (hepta-, octa-, nona-CHBs) Internal standard PCB-204... 

Quantification of analytes can be performed by the internal standard method, using an isotopically labeled internal standard added to the sample in a precise amount at the early stage of the analytical treatment. By selectively monitoring specific ions of the molecule to be quantified and the corresponding shifted ions of the labeled homologue as reference signal, the precision of the analytical results of GC-MS can be improved. Moreover, random errors during different steps of the sample preparation are minimized. This so-called isotopic dilution technique has been widely used in numerous fields. Several examples of this are reported below. 

Liu and Ding (2004) developed a method to separate simultaneously four homologues of alkyltrimethylammonium and four of dialkyldimethylammonium with internal standard (octylammonium) and using indirect UV detection. Decylbenzyldimethyl ammonium chloride was used as chromophore and the influence on separation of buffer concentration, pH and organic solvent (methanol and tetrahydrofuran) was studied. Besides, methanol concentration in a sample solution was tested to avoid the formation of micelles and surfactant adsorption onto the capillary. The method was applied to determine cationic surfactants in five hair conditioners, finding only four homologous (Ci2 ig) trimethylam-monium at a total concentration of between 0.14 and 2.15%. In one of them, analysis with... 

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