Protein precipitation matrix effects

As a general rule, APCI is less likely to demonstrate matrix effects and ESI is more likely to be affected by matrix effects. Sample clean-up is another important factor—protein precipitation is more likely to result in matrix effects than is solid phase extraction. Matrix effects may be caused by sample constituents that are not parts of the biological matrix. Mei et al.126 129 showed that certain brands of sample tube containers can produce matrix effects. They also demonstrated that Li-heparin, a common anticoagulant for plasma samples, can produce significant matrix effects... 

Dams et al. [18] developed a validated quantitative LC-APCI-MS-MS method for simultaneous determination of multiple illicit drugs and their metabolites in oral fluid. This substrate is being increasingly popular for forensic applications as it provides information on recent use, similarly to blood plasma/serum, although it can be obtained with a simple, noninvasive, collection. Sample pretreatment, though limited to protein precipitation with acetonitrile, was sufficient to avoid matrix effect (see Figure 20.2). 

However, protein precipitation usually produces severe matrix effect [54-56], Therefore, in most LC-MS published methods, more extensive sample extraction procedures were used, being liquid-liquid (LLE) and SPE the most frequent techniques. 

A simple PPT was developed for plasma and OFs recoveries of analytes from OF are often higher when compared to plasma, but sonication before precipitation has been proven necessary to help the analytes to release the protein binding, also in OF. Developing a multiclass method with PPT has proved to be simpler in respect to other extractive procedures, so it has been possible to obtain recoveries averaging 100 % and minimized matrix effects by the use of a deuterated ISTDs for each class of substances [93],... 

Hou W, Watters JW, McLeod HL (2004) Simple and rapid docetaxel assay in human plasma by protein precipitation and high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B 804 263-267 Schuhmacher J, Zimmer D, Tesche F, Pickard V (2003) Matrix effects during analysis of plasma samples by electrospray and atmospheric pressure chemical ionization mass spectrometry practical approaches to their elimination. Rapid Communications in Mass Spectrometry 17 1950-1957 Shah PW (2001) Guidance for Industry Bioanalytical Method Validation U.S. Department of Health and Human Services, Food and Drug Administration... 

A number of reports in the literature indicate that matrix effect is dependent on ionization type, sample preparation, and bio-fluid type [104,108,114, 116-119]. In 2003, Dams et al. [119] demonstrated that the APCI was less susceptible to matrix effect, thereby allowing for simplification of sample preparation prior to LC/MS/MS analysis without jeopardizing the quality of quantitative data, as shown in Table 7-8. More recently, Souverain et al. [117] investigated the matrix effect in bio-analysis of illicit drugs with both LC/ESI-MS/MS and LC/APCI-MS/MS. Four procedures—liquid-liquid extraction (LLE), SPE, protein precipitation (PP) with acetonitrile (ACN), and protein precipitation with perchloric acid (PA)— were tested to evaluate... 

The post-colunm infusion setup has been widely applied in matrix effects studies, e.g., in developing methods for the quantitative screening of drag discovery compoimds by fast-gradient (1 min ran time) LC-MS [83-85], in the optimization of the sample pretreatment in the analysis of methadone, comparing four off-hne and three on-hne sample pretreatment methods [77], and in evaluating various protein precipitating additives [86],... 

Matrix effects experienced in the analysis of microsomal incubation products (Ch. 10.6.1) were evaluated by Zheng et al. [92]. The individual effects of the Tris buffer, NADPH, and the microsomes on the ESI-MS response of 27 different drags were investigated in direct injection MS-MS experiments. The more polar analytes showed up to 5-fold ion suppression. Therefore, an automated Oasis-HLB SPE procedure was developed in 96-well plate format. Direct injection of protein-precipitated incubations yielded similar results. Additional use of fast LC separation prior to MS-MS analysis gave no further improvement. 

The effectiveness of various protein precipitation additives in terms of protein removal and matric effects was investigated [86]. Acetorritrile, trichloroacetic acid (TCA), and zinc sulfate were formd most effective in removing proteirrs (applied in a 2 1 additive-to-plasma ratio). By a post-colunm infusion setup (Figme 11.6), these three methods were further evaluated with respect to matrix effect for five different mobile-phase compositions. As both buffered, acidified, and pure methanol-water mobile phases were compared, actual conclusions are difficult. In the pure methanol-water mobile phases, the use of TCA enhances the response, probably by generating acidic conditions more favourable in ESI-MS. With buffered or acidified mobile phases, the difference in matrix effects between acetonitrile or TCA as protein precipitation additive was less pronounced. A similar comparison between acetonitrile, perchloric acid, and TCA as protein precipitation additives was reported by others [100]. With acid precipitation, lower analyte recoveiy and higher %RSD was observed. Therefore, precipitation by acetonitrile was preferred. 

Matrix components are not efficiently removed with protein precipitation and will be contained in the isolated supernatant or filtrate. In MS/MS detection systems, matrix contaminants have been shown to reduce the efficiency of the ionization process with atmospheric-pressure ionization (API) techniques [3-12]. The observation seen is a loss in response, and this phenomenon is referred to as ionization suppression. This effect can lead to decreased reproducibility and accuracy for an assay and failure to reach the desired limit of quantitation. Additionally, the efficiency of protein removal witli organic solvents is not complete and typically ranges from 98.7% to 99.8% [2] to leave residual amounts of protein that carry over into the analytical system and foul the ionization source of a mass spectrometer after repeated injections. 

The present authors have developed a procedure which minimizes any potential matrix effects by protein precipitation (Brown et al., 1984). This protein precipitation technique was originally developed for serum nickel (Sunderman et al., 1984) and markedly reduces matrix effects in the final atomic spectroscopic analysis. Briefly the procedure is as follows ... 

Filtration and injection or protein precipitation with acetonitrile and injection of the supernatant (the so-called dilute and shoot strategy) can provide a direct means to introduce samples into HPLC (41). However, the lack of a concentration step may limit the detection of some of the most potent drugs, while the absence of a purification step may favor matrix effects, hence false negative results. 

Sample preparation consisting of a protein precipitation step affords some degree of cleanup, but often this is insufficient to eliminate matrix effects, especially when extracts are analyzed using instruments that operate in the ESI mode [110]. Instruments operating in the APCl mode are generally less susceptible to matrix effects [92],... 

Figure 13.13. The continuous trace is an ESI matrix effect chromatogram of a 10- iL injection of protein-precipitated plasma while post-column infusing beclomethazone. The dashed chromatogram is an injection of standard beclomethazone and metabolite superimposed to indicate retention times of the analytes. MRM data 466 376, 2.1 mm x 5 cm CIS column, 200 pL/min, isocratic conditions 60740 CH3CN/H2O 2 mM NH4OAC.

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