Plasma extraction

Figure 5.67 Reconstructed ion chromatograms for Idoxifene and internal standard (ds-Idoxifene using LC-ToF-MS for (a) double-blank human plasma extract, (b) extract of blank human plasma containing internal standard (IS), and (c) control-blank human plasma spiked with Idoxifene at 5 gml , the LOQ of the method. Reprinted from 7. Chromatogr., B, 757, Comparison between liquid chromatography-time-of-flight mass spectrometry and selected-reaction monitoring liquid chromatography-mass spectrometry for quantitative determination of Idoxifene in human plasma , Zhang, H. and Henion, J., 151-159, Copyright (2001), with permission from Elsevier Science.

Figure 5. Chromatograms for theophylline in plasma extracts. Arrow indicates tneophyUine peak. Conditions 50 cm X 3 mm (i.d.) column with 10 fjm silica gel (Micropak Si 10 Varian) mobile phase, 84/15/1 chloroform/isopropanol/acetic acid flow rate, 40 rm/hr detector, UV,273nm(40).

FIGURE 4.2 (A) Chromatographic profile from extracts for an Abbott compound. The detection was UV at 205 nm. The identity of tracings (from bottom) are a reference standard, a blank plasma extract, a low limit of quantitation sample, and a dosed sample (12 hour) from a study subject. A run time of 13 minutes and a liquid-liquid extraction with back extraction is required for a rugged assay. (B) LC/MS tracing of a dosed sample (not the same sample). A 2.5 minute run time is sufficient for the assay. 

Matrix effect is a phrase normally used to describe the effect of some portion of a sample matrix that causes erroneous assay results if care is not taken to avoid the problem or correct for it by some mechanism. The most common matrix effects are those that result in ion suppression and subsequent false negative results. Ion enhancement may lead to false positive results.126 127 Several reports about matrix effects include suggestions on what can cause them and how to avoid them.126-147 While various ways to detect matrix effects have been reported, Matuszewski et al.140 described a clear way to measure the matrix effect (ME) for an analyte, recovery (RE) from the extraction procedure, and overall process efficiency (PE) of a procedure. Their method is to prepare three sets of samples and assay them using the planned HPLC/MS/MS method. The first set is the neat solution standards diluted into the mobile phase before injection to obtain the A results. The second set is the analyte spiked into the blank plasma extract (after extraction) to obtain the B results. The third set is the analyte spiked into the blank plasma before the extraction step (C results) these samples are extracted and assayed along with the two other sets. The three data sets allow for the following calculations ... 

To improve chromatographic separation, another analytical column could be used in addition to the monolith (Xu et al. 2006). The monolith column served as an extraction column only. Hsieh et al. (2000, 2002) utilized a polymer-coated mixed function (PCMF) Capcell C8 column (4.6 x 50 mm, Phenomenex) to provide dual functions—online plasma extraction and analyte separation. The silica was coated with a polymer containing both hydrophilic polyoxythylene and hydrophobic groups. The diluted plasma samples (1 1 to 1 3) were injected directly. No column deterioration was observed after 200 injections. 

Plasma and uri ne (di sulfoton only) Plasma extraction with ethyl acetate urine adjustment to pH 7.4, centrifugation, extraction with ethyl acetate Capillary GC/MS (SIM) No data >75 (urine) <10 (blood) Singh et al. 1986... 

Plasma Extract denatured sample with petroleum ether- GC-ECD 1.0 g/L 102 (for hexa) Willet et al. 1978... 

Selected ion monitoring gas chromatogram of plasma extract showing traces of siloxanes 5 years after 5-year-old breast implants were removed. [From D Flassbeck. B. Pffeiderer, R. Grumping, and A. V. Hirner,... 

The sensitivity of the LTQ-Orbitrap is demonstrated using a mixture of synthetic standards of buspirone metabolites spiked into a rat plasma extract. The samples were analyzed with a standard 4.6-mm HPLC column. LC-MS/MS chromatograms for the five-component mixture obtained using the Orbitrap and the LTQ mass spectrometers are compared in Figs 5.8a and h. Also shown are MS/MS (m/z 402) spectra of the oxa-buspirone metabolite (R, = 7.4 min) at 10 pg on column in the Orbitrap (Fig. 5.8c) and LTQ (Fig. 5.8e). As discussed above, the MS/MS fragmentation spectra, obtained in the Orbitrap (Figs 5.8c and d), are very similar to those from the LTQ (Fig. 5.8e), and even at such low concentrations excellent mass accuracies are maintained. 

Although the advantages associated with online plasma extraction are attractive, care must be taken to monitor the recovery of dmg-related material during the extraction process. Unlike quantitative plasma analysis, where poor recovery only affects the limit of quantitation, the recovery of all drug-related components in metabolite profiling studies must be high in order to ensure that the quantitative data are meaningful. 

To demonstrate that the proposed methods are suitable for structural elucidation of isomeric metabolites and derivatives in biological matrices, human plasma was spiked with the mixture of DL, 6-OH-DL, 3-OH-DL, /V-OH-DL, and 1-pyridine-/V-oxide-DL. The resulting sample was extracted and analyzed by LC-MS and LC-MS/MS in ESI and APCI modes as described above. HDX was successfully performed online when the extract was injected directly onto the HPLC column without drying and reconstituting the sample in a deuterated solvent. In general, there were no differences between the results obtained for the spiked plasma extract and for the mixture of the standard compounds, which indicates that the LC-MS methods with HDX described here are applicable for the analysis drug-derived material in plasma or other biological matrices. 

Sampson, D. Harasymiv, I., and Hensley, W.J. Gas chromatographic assay of underivatized 5,5-d iphenylhy-dantoin (Dilantin ) in plasma extracts. Clin. Chem. 17 382-385, 1971. 

Figure 6.41 Diagram of the instrument configuration for integrated dual BioTrap column plasma extraction and LC/MS analysis. Extraction proceeds on one column while the other column is equilibrating and rinsing. (Reprinted with permission from Needham et al., 1998. Copyright 1998 Elsevier.)...

Rat plasma Extraction/protein precipitation with acetonitrile UHPLC (C8) MS ESI hybrid quadrupole time of flight 12 (17)... 

The method of Cosyns et al [28] was modified with fluorescence detection to increase sensitivity [18]. The plasma extraction procedure and HPLC... 

Figure 8.10 Separation of benzodiazepines in plasma extracts using (a) conventional HPLC (4.6 mm I.D.) and (b) capillary LC (320 /rm I.D.). Conditions mobile phase, 35 10 55 (v/v/v) acetonitrile/methanol/10 mM phosphate buffer (pH 7.0) flow rate, 6.0 /d/min injection volume, 20 /d detection, UV absorbance at 313 nm. Peaks 1, carbamazepine 2, nitrazepam 3, clonazepam 4, flunitrazepam (internal standard) 5, nordazepam 6, diazepam. (Reprinted from Ref. 31 with permission.)...

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...

Different reversed phase [195,239,240], mixed mode (ion exchange and reversed phase) SPE cartridges [173,218] and online SPE column [193, 238] have been also reported for samples preparation and extraction. Some of these assays combined both PP and SPE in order to achieve an extensive sample cleanup [193, 195, 238-240], Likewise SPE, LLE provides cleaner plasma extracts than PP. Nevertheless, LLE procedure does not always provide satisfactory results with regard to extraction recovery and selectivity, especially with polar analytes and particularly in the case of multicomponent analysis such as in drug-metabolism studies, where analytes polarity varies widely. This issue was addressed by Zweigenbaum J and Henion J [235] and extraction solvent optimization, using isoamyl alcohol, to achieve acceptable extraction selectivity and recovery for polar analytes has been discussed. 

Blood plasma Extract with acetonitrile and hexane GC/MS 0.15 g/mL 93 Sjoberg and Bondesson 1985... 

The normal phase HPLC (20% chloroform in heptane) could separate A9-tetrahydrocannabinol from monohydrox-ylated metabolites and from 11-hydroxy-A tetrahydrocannabinol. However, a minor overlap could be avoided by collecting the tetrahydrocannabinol 1 in a slightly narrower volume range. The prior heptane extraction of alkalinized plasma had separated these non-polar constituents from any acidic metabolite. This separation of plasma extracts and normal phase HPLC collection of volumes in the appropriate range resulted in a substantial reduction in GLC background from plasma components for derivatized tetrahydrocannabinol analyzed with electron capture (63 1) detection. 

Figure 3. Mass fragmentograms of Cs -THC-TMS (m/e 386) with A6-THC-d3-TMS as internal standard (m/e 389) from purified plasma extracts. Plasma levels of 0.0,0.2,6.6, and 19 ng/mL.

Both normal phase and reverse phase HPLC methods were studied as analytical techniques for analysis of I and VI in human plasma. Normal phase was found to be more satisfactory for separation of I from plasma constituents whereas reverse phase was the choice for VI. Although reverse phase HPLC could be used to simultaneously assay for both I and VI when placed directly on the instrument, it was not practical for analysis of plasma extracts. 

The calibration standards representing LLOQ, ULOQ, and a midpoint serve as quality control samples. These standards are freshly prepared in control plasma, extracted, and run in parallel to the all gels. 

Add 2 ml of a saturated solution of borax to 2 ml of plasma, extract with 4 ml of toluene, separate the... 

Figure 13-4. Representative MRM scans (plasma extract of a proprietary compound) using an API 5000 triple quadrupole unit (Sciex). Each panel contains a distinct MRM transition for the same compound m/z 1021.6 —> 1003.5 (left panel) and m/z 1021.6 971.5 (right panel). Signal-to-noise ratio is designated as S/N. Experimental conditions ESI, positive ion mode, protein precipitation was used for sample preparation, injection volume was 10 pL, the column was a Cig, and dimension was 20 x 2.1 mm, using a linear gradient elution Omin (20% B)-6min (90% B)-8min (90% B), where B was 0.2% formic acid in acetonitrile and A was 0.2% formic acid in water separation was performed at room temperature.

C Sameridine 3 Batch Radioligand Off-line Plasma Extraction in heptane Heptane EtOH Heptane/ GC-FID [23]... 

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