Protein precipitation automated

Protein precipitation by filtration in a 96-well format has been used as a high-throughput, easy-to-automate alternative to the traditional centrifugation-based protocol. However, most filter plates... 

Conditions for Automated Solid Phase Extraction of Vitamin D Metabolite from Plasma after Protein Precipitation... 

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... 

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. 

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. 

The use of organic solvents may constitute a matrix compatible to subsequent liquid chromatography, thus not requiring any concentration or evaporation step. However, protein precipitation seems to be inappropriate for automation and thus requires a manual workflow. 

An example for an automated stability test in plasma is described by Linget and du Vignaud (1999). Incubations are performed on a 215 Gilson liquid handler. Incubation was done at substrate concentrations of 50 pM on 96 deep well plates. Each incubation tube contained 375 pL of a 200 pM test compound solution (in 0.1 M Tris buffer with 3% BSA, added to assist dissolution of compounds with poor solubility) and 1125 pL of plasma. Samples are taken after incubation times of 0, 1, 2, 3, 4 and 5 min. At each of these time points an aliquot of the incubation mixture was transferred from the incubation tube into a well in a 96 deep well plate containing an equal volume of acetonitrile for quenching by protein precipitation followed by centrifugation of the plates. Supernatants were analyzed by HPLC for metabolic screening. 

A. P. Watt, D. Morrison, K. L. Locker, and D. C. Evans, Higher throughput bioanalysis by automation of a protein precipitation assay using a 96-well format with detection by LC-MS/MS, AnaZ. Chem. 72 (2000), 979-984. 

T. Pereira and S. W. Chang, Semi-automated quantification of ivermectin in rat and human plasma using protein precipitation and filtration with liquid chro-matography/tandem mass spectrometry, Rapid Commun. Mass Spectrom. 18 (2004), 1265-1276. 

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. 

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. 

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]. 

R.A. Biddlecombe, S. Pleasance, Automated protein precipitation by filtration in the 96-well format, J. Chromatogr. B, 734 (1999) 257. 

D. O Connor, D.E. Clarke, D. Morrison, A.P. Watt, Determination of drug concentrations in plasma by a highly automated, generic and flexible protein precipitation and LC-MS-MS method applicable to the drug discovery environment. Rapid Commun. Mass Spectrom., 16 (2002) 1065. 

Certain techniques are very good at particular tasks (such as protein precipitation for removal of proteins and cellular components), but perhaps not as good in general. With the emphasis on greater efficiency, many of these approaches have become automated or semiautomated. Thus, there has been greater emphasis on the 96-well formats an approach that can lend itself more readily to automated workstations. This format has been used successfully for protein precipitation, liquid—liquid extraction, and solid-phase extraction [8-10]. Ultrafiltration (UF) in a 96-well format is also being evaluated and shows some potential, but products and applications are not yet fully developed. Automated techniques for sample preparation and each of the sample preparation techniques listed in Table 1 are described below. 

Recently, automated sample preparation approaches utilizing parallel sample processing have been described for SPE [10], LLE [9], simple dilutions [11], and protein precipitation [8]. These procedures utilize commercially available workstations for liquid handling in a 96-well multichannel plate format. These workstations are evolving rapidly and are constandy gaining additional capabilities. A recent article has reviewed the most common types and describes the major advantages of each [36]. 

The three main formats for sample preparation used in drug-discovery are protein precipitation (PPT), SPE, and LLE. Several examples of off-line sample preparation have been reported and involve SPE [37,38,46,47], LLE [38,48], and PPT [39,49]. In each of the examples cited, semi- or fully automated strategies for liquid handling were incorporated to enhance throughput. Even with the recent popularity of on-line methods, off-line techniques continue to be widely employed. The key advantage to off-line methods is that sample preparation may be independently optimized from the mass spectrometer and does not contribute overhead to the LC-MS injection duty cycle. 

A Unal example of direct bioanalysis was recently published by Dethy et al. and involves the appUcation of infusion nanoelectrospray (nano-ESI) from a silicon chip [110]. In this example, supernatant obtained from protein precipitation was directly infused with an automated pipette-tip delivery system. Individual, conductive pipette tips that contain sample were sequentially introduced to the backplane of a silicon chip for analysis. The front plane of the chip that consisted of 100 individual nano-ESI nozzles, was positioned near the API orifice of a TQMS for direct serial analysis. Quantitation of verapamil and its metaboUte norverapamil occurred in human plasma over a range of 5-1000 ng/mL. It is possible to achieve analysis times of less than 1 minute per sample with this technology. An important advantage, demonstrated by this work, is the unique abiUty to avoid system carryover with this device [110]. 

Watt, A.P Morrison, D. Locker, K.L. Evans, D.G. Higher Throughput Bioanalysis by Automation of a Protein Precipitation Assay Using 96-Well Format with Detection by LC-MS/MS, Anal. Chem. 72, 979-984 (2000). 

Biddlecombe, R.A. Pleasance, S. Automated Protein Precipitation by Filtration in the 96-Well Format, J. Chromatogr. B Biomed. Sci. Appl. 12,257-265 (1999). 

Biological matrices are not directly compatible with LC-MS analysis, since these samples tend to block LC columns and contaminate the ion source. Extraction of compounds of interest from biological fluids is required prior to LC-MS/MS analysis [20]. Sample extraction can be achieved off-line with protein precipitation (PP), liquid-liquid extraction (LLE), or solid-phase extraction (SPE) [21]. With the ease of use and sophistication of automated liquid-handling systems, sample extraction procedures in a 96-well format can handle microliter volumes with multiple sorbents per plate and can simplify and expedite SPE method development [22,23]. The technique can be used to routinely develop methods for multiple analytes and examine a set of eluent compositions for each analyte [16]. 

Each of these approaches is practical for performing high throughput protein-precipitation methods. The determining factors for selection of the collection-plate format over the filtration plate, or vice versa, include the extent of available hardware and automation accommodating the microplate format, total cost of materials, and thus the cost per sample, number of physical manipulations, and the degree of transfer loss deemed acceptable. 

Zhang, L. Laycock, J.D. Hayos, J. Flynn, J. Yesionek, G. Miller, K.J. Automated Strategies for Protein Precipitation Filtration and Solid Phase Extraction (SPE) Optimization on the TECAN Robotic Sample Processor—Applications in Quantitative LC-MS/MS Bioanalysis, paper presented at LabAutomation 2004, February 1-5,2004, San Jose, CA. 

---