Bioanalysis is quantitative detection of fraction of xenobiotic in the biological fluids. During the last 15 years much advancement have been made in domains of detection separation, samples pretreatment and data handling . Advances in bioanalysis do not usually come as a sudden revolution but by steady improvements and continuous progress on different fronts by different groups.
Liquid chromatography linked to the Mass Spectrometry (MS) has played a valuable role in the Pharmacokinetics and Metabolic studies at various stages since its introduction to the pharma industry.
Before introducing to the MS, Samples Pretreatment (SP) is the important and critical stage of the method development .As we all know that biological fluids consist of several complex proteins and other continuants in addition to the analyte to the interest . SP involves removal of unwanted matrix constituents and preconcentraion of analyte of interest in to a medium which can be injected /subjected to chromatographic separation and detection with greatest ease. And also SP should be based on thorough review of the xenobiotics physical and chemical properties .It should be systematic and once finalized must be tested for its ruggedness.
SP comprises mainly of Protein Precipitation (PP), Liquid Liquid Extraction (LLE) and Solid Phase Extraction (SPE) .PP is precipitation of protein by addition of denaturing Solvents/solution .Advantage are that it is easy to automate, lesser steps involved and cost it is effective. Howeverr it has a major limitation as many endogenous material also get extracted with the with the interest of the analyte which further requires extensive chromatographicoptimization. Moreover it can also impact the longevity of the method.
LLE has a wide variety of solvents options and pH adjustments is best and extensively used in SP techniques. Many organic solutions and solvents are used for the extraction of analyte of interest from the biological matrix (urine, blood, plasma, serum).As these solvents cannot be directly injectable to the chromatographic system ,a evaporation step is involved ,than samples into in aqueous based diluents which is very much compatible to the chromatographic system.
SPE based on basic principles of selective retention of analyte on to stationary bed .For this several specific and mixed mode stationary packing are available in the market ,but due to diverse nature of proposed application it is sometimes difficult to choose a stationary phase which is sufficient for all needs of application.
Sample preparation formatted to 96 well plates allows semi automation of offline sample preparation techniques significantly increasing throughput .Online SPE using column switching tech is rapidly gaining acceptance in bioanalysis to reduce both time and labour requirement to produce bioanalysis.
After sample pre-treatments , the sample is subjected to chromatographic separation . Newly introduced techniques such as UPLC with small particle size(2µ) and monolithic chromatography offers improvements in resolution and sensitivity compared to the conventional chromatographic techniques .Hydrophilic interaction chromatography(HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to conventional RP (reverse phase) application.
Particle base chromatographic has limitation of high back pressure which leads to frequent leakage and sudden batch stoppages in HPLC. The problems can be controlled with use of SS tubing and frequent flushing of solvents lines.
In current advanced techniques such as MEPS, micro extraction by packed sorbets is a new technique in sample preparation that can be connected on-line to gas chromatography (GC) or Liquid Chromatography (LC) without any modification . MEPS is a miniaturized ,SPE techniques that works with sample volumes as small as 10µL The key aspects of MEPS is that the solvent volume used for the elution of the analytes is of a suitable order of magnitude to be injected directly into GC or LC systems. It is useful for clinical and preclinical studies.
Detection is the important and final step of bioanalysis .Mass Spectrometry is filtering the ionized molecules through a electronic /magnetic field ,based on their mass to charge ratio. Recent advancements in field of Mass Spectrometry have uncompared selectivity and sensitivity over its other counterparts .Linear rails used in conventional application ,modified rail arrange Quardropole Traps having advantage to trap the ions inside the Q and enhance the selectivity and sensitivity. Senstivity of these instruments is in pg/mL levels for analytes and also selectivity of techniques to separate out the signal for analyte of interest has definitely given an edge to the bioanalytical scientists.
Future is unpredictable but still looking at the past use it can be presumed that improvements are the key for good quality and implementation of any new techniques to lab or application should be explored with same intention.
-The author is Senior Research Scientist (Bioanalytical),Semler Research Center, Bangalore |