Protein precipitation procedure

Typical protein precipitation procedures use one volume of plasma plus three to six volumes of acetonitrile or methanol (or a mixture) with the internal standard at an appropriate concentration for the assay. Poison et al.102 reported that protein precipitation using acetonitrile eliminates at least 95% of the proteins after filtration or centrifugation, the supernatant can often be directly injected into the HPLC/MS/MS system. Usually this step is performed using 96-well plates that are ideal for semi-automation of sample preparation. Briem et al.103 reported on a robotic sample preparation system for plasma based on a protein precipitation step and a robotic liquid handling system that increased throughput by a factor of four compared to a manual system. 

Protein Precipitation Procedures involving protein precipitation with trichloroacetic acid for Cu, Fe, and Zn have been developed for flame atomic absorption spectrometry (FAAS) determinations in body fluids [20, 21]. The physical separation of caseins and fat from milk whey can also be achieved using centrifugation at 30,000g. 

Protein preeipitation was automated into a 96-well plate format by means of a robotic liquid handler by Watt et al. [22]. Plasma samples (50 pi) were transferred from a 96-rack of tubes to a 96-well plate by means of a single-dispense tool. Acetonitrile (200 pi) was added to the wells by means of an 8-ohannel tool. The plate was removed, heat sealed, vortex-mixed for 20 s, and eentrifuged (2000g for 15 min). Using the 8 ehannel tool, the supernatant was transferred to a elean plate, to which first 50 pi of a 25 mmol/1 ammonium formate buffer solution was added. The plate is then removed, heat-sealed, vortex-mixed, and transferred to the autosampler for LC-MS analysis. The proeedure takes 2 hr per plate. A fourfold improvement in sample throughput on the LC-MS instrument was achieved, compared to previous manual protein precipitation procedures. 

Protein precipitation was automated into a 96-well plate format by means a robotic liquid handler by Watt et al. [101]. The procedure is described in more detail in Ch. 1.5.1. It enabled a 4-fold improvement in sample throughput on the LC-MS instrument, compared to previous manual protein precipitation procedures. The method was applied as a generic sample pretreatment method in combination with a generic LC-MS method to a variety of drug candidates [102]. 

Although many individual choices for sample preparation exist, sometimes the combination of two techniques yields a more desirable result. Within a drug discovery environment, however, throughput and cost are important factors that influence the choice of sample-preparation methods. The protein-precipitation procedure can quickly yield a sample that is ready for analysis, but its potential for carryover of matrix interferences is problematic. The isolated supernatant can be filtered or centrifuged before injection, but these procedures remove only particulates or proteinaceous material, not the materials that cause matrix interferences. Thus, protein precipitation is sometimes followed by LLE, SPE, or an on-line technique for a more selective cleanup before analysis. A particularly challenging sample-preparation requirement is the analysis of animal tissues, as is commonly performed in drug-discovery support laboratories that perform drug uptake studies. Here, a series of sequential sample-preparation steps are common [121]. 

Siegers, C.R Moller-Hartmann, W. Cholestyramine as an antidote against paracetamol-inducedhepato-and nephrotoxicity in the rat. Toxicol.Lett., 1989, 47, 179-184 [rat urine extracted metabolites] Lam, S. Malikin, G. An improved micro-scale protein precipitation procedure for HPLC assay of therapeutic drugs in serum. J.Liq.Chromatogr., 1989, 12, 1851-1872 [serum also amiodarone, aspirin, caffeine, chloramphenicol, flecainide, pentobarbital, procainamide, pyrimethamine, quinidine, theophylline, tocainide, trazodone fluorescence detection UV detection]... 

Preparation of fiiiit juices and pharmaceutical samples prior to TLC presents no problems, but for saipples containing protein, precipitation procedures must be used first (Bui-Nguyen, 1985). For instance, serum samples must be deprotei-nized by adding two volumes of methanol or acetonitrile for each volume of serum. 

Some flavin enzymes may be separated into the apoenzyme and coenzyme components by a shift of pH and dialysis, or protein precipitation procedures. The old yellow enzyme was the flrst example of this, in that the active enzyme protein could be regenerated from coenzyme and apoenzyme (Theorell, 1934). But the bonds often are much tighter, and the flavin groups generally do not dissociate from the protein. According to our definition, they are prosthetic groups. Some flavoproteins also contain tightly bound metal ions which probably participate in catalysis (cf. Chapt. X-4). 

Many use the hexokinase procedure without protein precipitation. That this is a procedure which is not acceptable is illustrated in Table II where it is shown that unless hemoglobin is removed there will be interference in the results obtained. In severely hemolyzed blood, errors as high as 25% are not uncommon. 

If the fluorometric procedure is used, with protein precipitation, then bilirubin will not interfere with the hexokinase procedure. Even if protein precipitation is not resorted to, the interference from bilirubin does not become significant until one is dealing with an infant in severe jaundice. This can be seen in Table III. 

If the fluorometric method is used after protein precipitation then the glucose can be readily assayed on 1 pi of plasma with the hexokinase procedure. 

Effect of bilirubin on the glucose fluorometric hexokinase procedure with and without protein precipitation, ... 

Protein extraction procedures employing chemicals such as detergents are effective in many instances, but they suffer from a number of drawbacks, not least of which is that they often induce protein denaturation and precipitation. This obviously limits their usefulness. Furthermore, even if the chemicals employed do not adversely affect the protein, their presence may adversely affect a subsequent purification step (e.g. the presence of detergent can prevent proteins from binding to a hydrophobic interaction column). In addition, the presence of such materials in the final preparation, even in trace quantities, may be unacceptable for medical reasons. 

FIGURE 1.39 Solvent-first procedure using ISOLUTE PPT Protein Precipitation Plates.159 (Reproduced with permission from Biotage AB.)... 

For discovery PK assays, the most common sample preparation procedure is protein precipitation161720 24 because it is fast, easy to automate, and requires no method development. While protein precipitation typically will not provide as clean a sample as will alternative procedures, it is sufficient for most discovery PK samples that use HPLC/MS/MS for the analytical step.21101... 

The most common (off-line) sample preparation procedures after protein precipitation are solid phase extraction and liquid-liquid extraction. Multiple vendors and available chemistries utilize 96-well plates for solid phase extraction systems and liquid-liquid extraction procedures. Both extraction process can prepare samples for HPLC/MS/MS assay. Jemal et al.110 compared liquid-liquid extraction in a 96-well plate to semi-automated solid phase extraction in a 96-well plate for a carboxylic acid containing analyte in a human plasma matrix and reported that both clean-up procedures worked well. Yang et al.111 112 described two validated methods for compounds in plasma using semi-automated 96-well plate solid phase extraction procedures. Zimmer et al.113 compared solid phase extraction and liquid-liquid extraction to a turbulent flow chromatography clean-up for two test compounds in plasma all three clean-up approaches led to HPLC/MS/MS assays that met GLP requirements. 

Direct injection of plasma or supernatant after protein precipitation on a short column with a high liquid flow rate is a common method for reducing analysis time in the pharmaceutical industry. The direct injection of a sample matrix is also known as the dilute-and-shoot (DAS) approach.62 DAS can be applied to all types of matrices and approaches and is the simplest sample preparation method with matrix dependency. Direct injection can also be approached through the extraction of eluent from PPT, SPE, and LLE onto a normal phase analytical column. The procedure is called hydrophilic interaction liquid chromatography (HILIC)70110111 and it avoids the evaporation and reconstitution steps that may cause loss of samples from heat degradation and absorption. 

Before compounds in biological matrices can be analyzed by LC/MS/MS, the samples must undergo a preparation procedure. There are a variety of techniques available for sample preparation including offline sample preparation techniques (liquid-liquid extraction, protein precipitation, and solid phase extraction) and on-line sample preparation... 

Marchi, I., Rudaz, S., Selman, M., and Veuthey, J. L. (2007). Evaluation of the influence of protein precipitation prior to on-line SPE-LC-API/MS procedures using multivariate data analysis.. ... 

Drug/Lab test interactions Because false-positive readings were reported with the Ames N-Muitistix SG dipstick test for urinary protein when gabapentin was added to other antiepileptic drugs, the more specific sulfosalicylic acid precipitation procedure is recommended to determine the presence of urine protein. 

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