Introduction:
LC-MS is used for the bioanalysis of small molecules drugs, which has molecular mass of less than 800 Da. Now a days, it is also frequently used for the bioanalytical determination of biopharmaceuticals.1 In pharmaceutical R & D, it takes a year to develop a new marketing drug identified from thousands of new compounds. The drug discovery is divided into early and late phase, during which pharmacokinetics study or bioanalysis of a drug is done. Once, the bioanalysis of a drug is done, the next step is the drug development in which, a drug is studied for its toxicokinetics during its pre-clinical and clinical trials. After all these trials get succeded in proving the drug safety and efficacy, it is approved by the FDA for marketing.
Most of the bioanalysis activities involved in the drug discovery and …show more content…
For larger peptides having molecular weight of 1.5 – 10 kDa, aqueous protein precipitation is done using TFA ot TCA upto 10%, followed by evaporation or solid phase extraction to remove the acid. PEGylated proteins of larger sizes, with molecular weight of 40-50 kDa, possess a unique hydrophobicity which allows their precipitation. The PPT step denatures the proteins and removes matrix effect causing componenets. The precipitated proteins are then digested using trypsin and the analysed using LC-MS.
• Solid phase extraction (SPE) :
This method is used for extraction of small peptides or digested peptides. This method commonly use the reversed phase SPE or ion exchange SPE. The later technique provides additional dimension of separation. This method uses size exclusion principle. For example, pore size of 40 – 80 A° can roughly eliminate proteins larger than 20 kDa. The sample is added in a pre-conditioned solution and then washed to remove the interferents present in the sample after which the sample is eluted.
• Abundant protein depletion
LC-MS is used for the bioanalysis of small molecules drugs, which has molecular mass of less than 800 Da. Now a days, it is also frequently used for the bioanalytical determination of biopharmaceuticals.1 In pharmaceutical R & D, it takes a year to develop a new marketing drug identified from thousands of new compounds. The drug discovery is divided into early and late phase, during which pharmacokinetics study or bioanalysis of a drug is done. Once, the bioanalysis of a drug is done, the next step is the drug development in which, a drug is studied for its toxicokinetics during its pre-clinical and clinical trials. After all these trials get succeded in proving the drug safety and efficacy, it is approved by the FDA for marketing.
Most of the bioanalysis activities involved in the drug discovery and …show more content…
For larger peptides having molecular weight of 1.5 – 10 kDa, aqueous protein precipitation is done using TFA ot TCA upto 10%, followed by evaporation or solid phase extraction to remove the acid. PEGylated proteins of larger sizes, with molecular weight of 40-50 kDa, possess a unique hydrophobicity which allows their precipitation. The PPT step denatures the proteins and removes matrix effect causing componenets. The precipitated proteins are then digested using trypsin and the analysed using LC-MS.
• Solid phase extraction (SPE) :
This method is used for extraction of small peptides or digested peptides. This method commonly use the reversed phase SPE or ion exchange SPE. The later technique provides additional dimension of separation. This method uses size exclusion principle. For example, pore size of 40 – 80 A° can roughly eliminate proteins larger than 20 kDa. The sample is added in a pre-conditioned solution and then washed to remove the interferents present in the sample after which the sample is eluted.
• Abundant protein depletion