About Authors:
*S.B.Muthu Vadivel, R.Suresh Kumar1, A.Tamil Selvan2, R.Suthakaran3
Department of Pharmaceutical Analysis and Quality Assurance
Teegala Ram Reddy College of Pharmacy
Saroor nagar, Meerpet, Hyderabad – 97.
*muthuvadivelanalyst@gmail.com
Abstract
Swift growth in the use of LC-MS/MS for the analysis of drugs in biological matrices has been compelled by the need for timely and high-quality data at many stages in drug discovery and development process: from high throughput screening of drug candidates and rapid data generation for pre-clinical studies to almost real-time analysis of clinical samples. Prompt and rational method development, validation, and transfer play a pivotal role in achieving the goals of faster, better, and cheaper for pharmacokinetic studies since this could easily account for more than 50% of the time and labor resources for a moderate-sized project. In this review article, Processing and treatment of biological samples for LC-MS/MS methods will be critically reviewed and discussed. Nature of biological samples, preliminary treatment, extraction procedures, and how chromatography coupled with mass spectroscopy just a few topics covered in this review. Other interesting approaches for improving the storage and stability of biological sample extracts as well as multiplexing of LC columns will also are discussed.
Reference Id: PHARMATUTOR-ART-1607
INTRODUCTION
1.0 BIOPHARMACEUTICAL ANALYSIS
1.1. NEED FOR BIOPHARMACEUTICAL ANALYSIS
Methods of measuring drugs in biologicalmedia are increasingly important related to following
* Bioavailability and Bioequivalence Studies
* New Drug Development
* Clinical Pharmacokinetics
* Research in Basic Biomedical and Pharmaceutical Sciences
1.2. ASSAY OF DRUGS AND THEIR METABOLITES1
A number of allusions have been made to methods that distinguish drugs from their metabolites. Drug metabolism reactions can be divided into phase I and phase II categories. Phase I typically involves oxidation, reduction, and hydrolysis reactions. In contrast, phase II transformations entail coupling or condensation of drugs. This involves glucoronidation, sulfation, aminoacid conjugation, acetylation and methylation. Except for reduction processes, most phase I and phase II reactions yield metabolites that are more polar and hence more water soluble than the parent drug. Assays must distinguish between drug and its metabolites. If this fact is ignored, erroneous data may be generated.
1.3. ANALYSIS OF DRUGS IN VARIOUS BIOLOGICAL MEDIA
The most common samples obtained for biopharmaceutical analysis are blood, plasma and urine. Faeces are also utilized, especially if the drug or metabolite is poorly absorbed or extensively excreted in the bile. Other media that can be utilized includes saliva and tissue.
The choice of sampling media is determined largely by the nature of the drug study. All most the drug levels in a clinical pharmacokinetic study demand the use of blood, urine and possibly saliva. A bioavailability study may require drug level data in blood and/or urine whereas a drug identification or drug abuse problem may be solved with any one type of biological sample.
Detection of a drug or its metabolite in biological media is usually complicated by the matrix. Because of this, various types of cleanup procedures involving techniques such as solvent extraction and chromatography are employed to effectively separate drug components from endogenous biologic material. The ultimate sensitivity and selectivity of the assay method may be limited by the efficiency of the cleanup methodology.
If the blood is allowed to clot and is then centrifuged, about 30 to 50% of the original volume is collected as serum (upper level). Thus, plasma generally is preferred because of its greater yield from blood. Blood, serum or plasma samples can be utilized for drug studies and may require protein denaturation steps before further manipulation.
If plasma or serum is used for the procedure, the fresh whole blood should be centrifuged immediately at 4000rpm for approximately 5 to 10 mints and the supernatant should be transferred by means of a suitable device, such as a Pasteur pipette, to a clean container of appropriate size for storage.
Urine is easiest to obtain from the patient and also permits collection of large and frequently more concentrated samples. The lack of protein in a healthy individual's urine obviates the need for denaturation steps. Because urine samples are readily obtained and often provide the greatest source of metabolites, they are frequently analyzed in drug metabolism studies.
With humans, faeces are collected in an aluminium foil pan placed under a toilet seat. Once collected, the foil is folded around the material and the sample lyophilized. Faecal specimens contain high protein content, and difficulties arise in their handling and analysis (even after Lyophilization) because of the large ratio of solid mass to drug. Denaturation of protein is usually required before further manipulations are begun.
Saliva and biological media obtained from humans when constant ratio between plasma and salivary levels of certain drugs exists via non invasive sampling techniques. Saliva is advantageous in drug studies done with children. Although the concentrations of drugs in saliva are rarely equal to those in plasma, a constant ratio (over an effective therapeutic range) permits calculation of plasma levels based on salivary analysis.
Separation or isolation of drugs and metabolites from biologic samples is performed in order to partially purify a sample. In this manner, an analyst can obtain the selectivity and sensitivity needed to detect a particular compound and can do so with minimum interference from components of the more complex biological matrix. The number of steps in a separation procedure should be kept to a minimum to prevent loss of drug or metabolite. Sometimes, the separation steps are preceded by a sample pretreatment.
1.4. STORAGE REQUIREMENTS FOR BIOLOGICAL SAMPLES
In order to avoid decomposition or other potential chemical changes in the drugs to be analyzed, biological samples should be frozen immediately upon collection and thawed before analysis. When drugs are susceptible to plasma esterase’s, the addition of esterase inhibitors, such as sodium fluoride, to blood samples immediately after collection helps to prevent drug decomposition.
When collecting and storing biological samples, the analyst should be wary of artifacts from tubing or storage vessels that can contaminate the sample. For example, plastic-ware frequently contains the high boiling liquid bis (2-ethylhexyl) phthalate; similarly, the plunger-plugs of vacutainers are known to contain tri-butoxyethyl phosphate, which can interfere in certain drug analysis.
1.5. PRELIMINARY TREATMENT OF BIOLOGICAL SAMPLES
In most cases, preliminary treatment of a sample is needed before the analyst can proceed to the measurement step. Analysis is required for drug in samples as diverse as plasma, urine, faeces, saliva, bile, sweat and seminal fluid. Each of these samples has its own set of factors that must be considered before an appropriate pretreatment method can be selected. Such factors as texture and chemical composition of the sample, degree of drug-protein binding, chemical stability of the drug and types of interferences can affect the final measurement step.
1.5.1. PROTEIN PRECIPITATION OR DENATURATION1, 2
Biological materials such as plasma, faeces, and saliva contain significant quantities of protein, which can bind a drug. The drug may have to be freed from protein before further manipulation. Protein denaturation is important, because the presence of proteins, lipids, salts, and other endogenous materials in the sample can cause rapid deterioration of HPLC columns and also interfere the assay.
Protein denaturation procedures include the use of tungstic acid, ammonium sulfate, heat, alcohol, trichloroacetic acid and perchloric acid.
Methanol and acetonitrile frequently have been used as protein denaturants of biological samples. Methanol sometimes is preferred because it produces a flocculent precipitate and not the gummy mass obtained with acetonitrile. Methanol also gives a clearer supernatant and may prevent the drug entrapment that can be observed after acetonitrile precipitation.
Ultra filtration and dialysis procedures also have been used to remove proteins from biological fluids. These procedures are not widely used because they are slow.
1.5.2. HYDROLYSIS OR CONJUGATES3
The presence-of drug metabolites as conjugates, such as glucuronides and sulfates, in biological samples cannot be ignored. The effect of a drug depends to a considerable extent on the biotransformation that occurs in the human body. Therefore, it may be important to isolate the actual conjugates. Samples containing either glucuronide acetals or sulfate esters are usually pretreated using enzymatic or acid hydrolysis. The unconjugated metabolites that result from the hydrolysis procedure are less hydrophilic than their conjugates and usually can be extracted from the biological matrix.
A nonspecific acid hydrolysis can be accomplished by heating a biological sample for 30 min at 90 to 100°C in 2 to 5N hydrochloric acid. Upon cooling, the pH of the sample can be adjusted to the desired level and the metabolite removed by solvent extraction. Particularly stable conjugates sometimes require hydrolysis in an autoclave.
1.5.3. HOMOGENIZATION
For samples containing insoluble protein, such as muscle or other related tissues, a homogenization or solubilizing step using 1N hydrochloric acid may be required before treating the sample further. For gelatinous samples such as seminal fluid or sputum, liquefaction is achieved via sonication. A solid sample such as faeces can be homogenized with a minimum amount of methanol. Homogenization is usually performed with a blade homogenizer (e.g., Waring Blender).
2.0. EXTRACTION PROCEDURES FOR DRUGS AND METABOLITES FROM BIOLOGICAL SAMPLES4, 5, 6
After pre treatment of biological material, the next step is usually the extraction of the drugs from the biological matrix. All separation procedures use one or more treatments of matrix-containing solute with some fluid. As extracting solvents are liquid and the biological sample solid (e.g., lyophilized faeces), it is an example of liquid-solid extraction. If the extraction involves two liquid phases, it is an example of liquid-liquid extraction.
2.1. LIQUID-SOLID EXTRACTION
Liquid - solid extractions occur between a solid phase and a liquid phase, either phase may initially contain the drug substance. Among the solids that have been used successfully in the extraction (usually via adsorption) of drugs from liquid samples are XAD-2 resin, charcoal, alumina, silica gel, and aluminum silicate. Sometimes the drugs are contained in a solid phase, such as in lyophilized specimens. Liquid-solid extraction is often particularly suitable for polar compounds that would otherwise tend to remain in the aqueous phase. The method could also be useful for amphoteric compounds that cannot be extracted easily from water.
Factors governing the adsorption and elution of drugs from the resin column include solvent polarity; flow rate of the solvent through the column and the degree of contact between the solvent and with the resin beds.
In the adsorption process, the hydrophobic portion of the solute that has little affinity for the water phase is preferentially adsorbed on the resin surface while the hydrophilic portion of the solute remains in the aqueous phase. Alteration in the lipophilic / hydrophilic balance within the solute or solvent mix and not within the resin, affects adsorption of the solute.
Biological samples can be prepared for cleanup by passing the sample through the resin bed where drug (metabolite) components are adsorbed and finally eluted with an appropriate solvent. The liquid-solid extraction method provides a convenient isolation procedure for blood samples, thus avoiding solvent extraction, protein precipitation, drug losses and emulsion formulation. It is possible; however, that strong drug-protein binding could prevent sufficient adsorption of the drug to resin.
2.2. DEHYDRATION METHODS.
An aqueous biological sample is treated with a sufficient quantity of anhydrous salt (sodium or magnesium sulfate) to create a "dried" mix. This mix is then extracted with a suitable organic solvent to remove the desired drug or metabolite.
2.3. LIQUID-LIQUID EXTRACTION
Liquid-liquid extraction is probably the most widely used technique because the analyst can remove a drug or metabolite from larger concentrations of endogenous materials that might interfere with the final analytical determination. The technique is simple, rapid and has a relatively small cost factor per sample.
The extract containing the drug can be evaporated to dryness and the residue can be reconstituted in a smaller volume of a more appropriate solvent. In this manner, the sample becomes more compatible with a particular analytical methodology in the measurement step, such as a mobile phase in LCMS/MS determinations.
The extracted material can be reconstituted in small volumes (e.g., 100 to 500 µl of solvent), thereby extending the sensitivity limits of an assay. It is possible to extract more than one sample concurrently. Quantitative recoveries (90% or better) of most drugs can be obtained through multiple or continuous extractions.
Partitioning or distribution of a drug between two possible liquid phases can be expressed in terms of a partition or distribution coefficient, usually called partition coefficient is constant only for a particular solute, temperature, and pair of solvents used. By knowing the P value for the extracted drug and the absolute volumes of the two phases to be utilized, the quantity of drug extracted after a single extraction can be obtained. In multiple extractions methodology, the original biological sample is extracted several times with fresh volumes of organic solvent until as much drug as possible is obtained. Because the combined extracts now contain the total extracted drug, it is desirable to calculate the number of extractions necessary to achieve maximum extraction.
2.3.1. FACTORS AFFECTING THE PARTITION COEFFICIENT8
Factors that influence partition coefficient and hence recovery of drugs in liquid-liquid extraction are choice of solvent, pH and ionic strength of the aqueous phase. In almost all cases, one of the liquid phases is aqueous because of the nature of a biological sample. The second liquid is selected by the analyst. It is highly desirable to select an organic solvent that shows greater affinity for the drug analyzed, yet leaves contaminants or impurities in the aqueous or biological phase. The solvent should be immiscible with an aqueous phase, should have less polarity than water, and should solubilize the desired extractable compound to a large extent. It should also have a relatively low boiling point so that it can be easily evaporated if necessary. Other considerations are cost, toxicity, flammability, and the nature of the solvent. If larger numbers of samples are to be extracted, the volume of solvent needed per sample can affect the overall cost of the assay procedure.
It is generally accepted that diethyl ether and chloroform are the solvents of choice for acidic and basic drugs, respectively, especially when the identity of the drugs in the samples are unknown. In these cases, any chemically neutral drugs are extracted into either solvent depending on their relative partition tendencies.
Proper pH adjustment of a biological sample permits quantitative conversion of an ionized drug to an un-ionized species, which is more soluble in a nonpolar solvent and therefore, extractable from an aqueous environment. In analysis, do determine a known drug or metabolite, the proper pH for extraction can be calculated from the Henderson-Hassel Balch equation using the pKa of the compound. If the species to be analyzed is unknown, the pH must be approximated based on the chemical nature of the suspected agent.
Third Factor influencing extractability of drugs from biological samples is ionic strength. Addition of highly water-soluble ionized salts, such as sodium chloride, to an aqueous phase creates a high degree of interaction between the water molecules and the inorganic ions in solution. Fewer water molecules are free to interact with an unionized drug. Therefore, the solubility of the drug in the aqueous phase decreases, thereby increasing the partitioning or distributing in favor of the non-polar or organic phase. The technique is commonly called "salting out."
Either mechanical or manual tumbling, rocking, or vigorous shaking of the samples can accomplish mixing of the aqueous organic phases. The percent recovery of a drug vs. time and/or type of mixing should be investigated for each biological sample. In many cases, vigorous shaking of a sample should be avoided because it leads to emulsification, which can be intractable for centrifugation. Emulsification is often observed when organic solvents are used at basic pH whereas certain organic solvents such as n-hexane and diethyl ether are less emulsion-prone.
Certain types of amphoteric drugs or drugs that possess extreme water solubility are not amenable to classic solvent extraction. In these cases, other types of analytical methodology such as ion-pairing must be adopted.
The technique of back-extraction can be applied with success to the analysis of drugs in biological samples. The purpose of the methodology is to further purify an extract by removing either drug or impurities by additional extractions.
3.0. CHROMATOGRAPHIC METHODS9
The presence of metabolites or more than one drug in a biological sample usually demands a more sophisticated separation for their measurement especially, when two or more drugs are of similar physical and chemical nature. Chromatography is a separation technique that is based on differing affinities of a mixture of solutes between at least two phases. The result is a physical separation of the mixture into its various components. The affinities or interactions can be classified in terms of a solute adhering to the surface of a polar solid (adsorption), a solute dissolving in a liquid (partition) and a solute passing through or impeded by a porous substance based on its molecular size (exclusion).
3.1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY9
HPLC is directly derived from classic column chromatography in that a liquid mobile phase is pumped under pressure rather than by gravity flow through a column filled with a stationary phase. This has resulted in a sharp reduction in separation time, narrower peak zones, and improved resolution. The mobile phase is placed in a solvent reservoir for pumping into the system. In the case of liquid-solid HPLC, solvents are chosen from the elutropic series. A solvent system is usually degassed by vacuum treatment or sonication before use.
3.2 LIQUID CHROMATOGRAPHY MASS SPECTROMETRY8, 9
Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for nonvolatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins.
Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-VIS) detectors. Some of these generate two dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode array UV-VIS detectors, generate three-dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time.
Mass spectrometers also generate three dimensional data. In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity and purity of a sample.
Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analysis.
For most compounds, a mass spectrometer is more sensitive and far more specific than all other LC detectors. It can analyze compounds that lack a suitable chromophore. It can also identify components in unresolved chromatographic peaks, reducing the need for perfect chromatography.
Some mass spectrometers have the ability to perform multiple steps of mass spectrometry on a single sample. They can generate a mass spectrum, select a specific ion from that spectrum, fragment the ion and generate another mass spectrum; repeating the entire cycle many times. Such mass spectrometers can literally deconstruct a complex molecule piece by piece until its structure is determined.
Mass spectral data complements data from other LC detectors. While two compounds may have similar UV spectra or similar mass spectra, it is uncommon for them to have both.
4.0. ESTIMATION OF DRUGS IN BIOLOGICAL SAMPLES BY LC-MS/MS10
MS has emerged as an ideal technique for the identification of such structurally diverse metabolites. When coupled with online HPLC the technique is extremely robust, rapid, sensitive, and easily automated. Not surprisingly, LC/MS/MS have become the methods of choice for pharmacokinetic studies, yielding concentration versus time data for drug compounds from in vivo samples such as plasma.
LC-MS instrument consist of three major components
· LC (to resolve a complex mixture of components)
· An interface (to transport the analyte in to the ion source) of a mass spectrometer
· Mass spectrometer (to ionize and mass analyze the individually resolved components)
Reverse phase (RP) HPLC is a widely pretended mode of chromatography and is a major contributing factor to advances made in several areas of pharmaceutical science. Mobile phase composition is a very critical in achieving selectivity in RP-HPLC separation. Although a large number of buffer system have been used in conventional RP-HPLC, only the volatile ion paring reagent can be used in LC-MS analysis.
REFERENCES
1. Guidance for Industry-Bioanalytical Method Validation, US Department of Health and Human services, Food and Drug Administration, Center for Drug and Evaluation and Research(CDER) and Center for Veterinary Medicine(CVM), (2001),http://fda.gov/cder/guidance/index.htm
2. Evans.G, Handbook of Bioanalysis and Drug metabolism,8-69,(2004)
3. W.M.A. Niessen, Liquid Chromatography-Mass spectrometry,2nd edition Vol-79,31-69,287-290,337-343,(1999)
4. Cole R, ESI-MS Fundamental instrumental method of chemical analysis.
5. Merrit W,Settle D Instrumental methods of Chemical analysis, 465, 502, 513,-529, 580-592,(2000)
6. Lee M, LC-MS fundamental Instrument and application, 3rd edition
7. Bonate p, Howard, Pharmacokinetic and drug development,2nd edition, 105,127,229, (2004)
8. Willard, Instrumental method of analysis, 6th edition.
9. Stavros K.HPLC Made to Measure A Practical Handbook for Optimization. WILEY-VCH Verlag GmbH & Co. KGaA; 2006:151-175,254.
10. Delatour C and Leclercq L. Positive electrospray liquid chromatography/ mass spectrometry using high-pH gradients: a way to combine selectivity and sensitivity for a large variety of drugs. Rapid Communications in Mass Spectrometry 2005; 19: 1359–1362.
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