Bioanalytical Techniques Revised.pptx

Bioanalytical Techniques Revised

1. Bioanalytical Techniques By Muhammad Rashad PhD Scholar (Pharmaceutical Chemistry) Biochemistry of Drug Metabolizing Enzyme

2. Drug metabolism involves a series of biological and chemical processes through which endogenous substances are converted into more water-soluble substituents resultantly excreted out from the body. Drug metabolic pathways accomplished by phase I and phase II biotransformation reactions. Drug Metabolism

3. Phase I reactions comprise oxidation, reduction, and hydrolysis. Phase II biotransformation reactions also called conjugation reaction includes glucuronidation, acetylation and sulfation reaction.

4. Models for drug metabolism evaluation Various in vitro models are developed for the drug metabolomics analysis, some of them are described below.

5. Models Human liver microsomes Human liver cytosol fractions S9 fractions Cell lines Transgenic cell line Isolated perfused liver Recombinant human CYP and UGT enzymes Hepatocytes Liver slices

6. Approaches for metabolite quantification Quantification of the metabolite is essential in such circumstances when metabolite concentration just reaches or crosses the limit of plasma drug concentration or when the drug metabolites are either pharmacologically active or toxic.

7. Direct quantification Drug metabolites are much more hydrophilic as compared to the parent drug, especially in case of glucuronides. for precise and definitive quantification of biological samples, appropriate authentic standards are required. Chemical synthesis is generally appropriate for making phase I metabolites, including O-demethylation, N-oxidation, N-demethylation, carbonyl reduction and others.

8. 2. Indirect quantification This technique has been employed for estimation of phase II metabolites especially glucuronide metabolites. These metabolites are detected by splitting the conjugates to produce original drug, which is then detected. In the case of morphine which is metabolized into two metabolite isomers, including morphine-3-glucuronide and morphine-6-glucuronide, former is an inactive metabolite while later possess more pharmacological activity even more than the parent drug. NMR based metabolomics mostly used for the indirect quantification of drug metabolites.

9. 3. Qualitative evaluation Mostly every type of mass spectrometer can be employed for the determination and recognition of glucuronide metabolites. However, those with tandem or high-resolution MS capacities are preferred. For the detection and validation of metabolites, LC/QTOF or LC/TOF instruments with tandem MS are commonly used. Currently, the use of Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICR / MS) and Ion Trap-Orbitrap (LTQ-Orbitrap) become more preferable tools in the metabolomics analysis as these have more compatibility with multiple LC methods.

10. Bioanalytic techniques for metabolites identification For the analysis of hundreds of metabolites, several analytical techniques are used. But none of the bioanalytical technique can fully analyze all metabolites in the sample due to their physicochemical diversity, including amino and non-amino organic acids, volatile ketones, alcohols, lipids, and carbohydrates, etc. Hence, different analytical techniques are used in combination with each other to fully measure the whole metabolomics.

11. Bioanalytical Techniques NMR GC-MS LC-MS/MS LC-MS However, NMR as compared to GC-MS and LC-MS less sensitive and more than 80% of metabolomics studies are carried out by using these two methods.

12. NMR based metabolite identification and quantification NMR coupled with MS provides optimum outcomes, but this technique is not commonly used because of its high cost. For the structural elucidation of organic compounds, NMR spectroscopy is frequently used. H1 NMR is a more useful and sensitive technique for the metabolites analytical study than C-NMR spectroscopy. The working principle behind NMR spectroscopy is the nuclei spinning of the atom, as metabolic components are made up of atoms and each atom has its nuclei.

13. H 1 NMR spectrum provide following pieces of information about the metabolites: The number of signals reflects the number of magnetically and chemically different protons. Such as the methanol molecule is comprised of methyl and hydroxyl (two sets of) protons, therefore in H1 NMR spectrum produce two signals. The area under the NMR peak refers to the intensity, which can be measured through the integrator. The relative position of NMR signals which is also known as the chemical shift, determined by special arrangement of adjacent electrons and atoms. The splitting of NMR signals determines the coupling constants.

14. The first step in NMR spectroscopy working is the sample preparation. Biological fluids including plasma require sample preparation for NMR but some biological fluids such as cerebrospinal fluids need no sample preparation. Tagging is the second step. Hydrogen atom attached to N-15 is used as an active label. The last step is quantification; a reference sample of known concentration must be added to determine the absolute concentration. Principle

15. One dimensional H1 NMR spectroscopy is a fast, reliable, and reproducible technique. 1D H1 NMR spectroscopy can identify and quantify 50-100 metabolites at a time. Moreover, 2D H1 NMR spectroscopy as compared to 1D H1 NMR can identify and quantify more metabolites. 13C NMR has advantageous resolution results than H1 NMR but due to the low natural abundance of 13C (∼1.1%) has less sensitivity towards the 13C nucleus, which significantly reduced 13C NMR applications in metabolomics. 15N NMR spectroscopy has wide applications in the structure elucidation and identification of DNA, RNA and proteins but owing to the low natural abundance of 15N (0.37%), not readily used in metabolomics studies. 31P nucleus has about 100 % relative abundance and a hundred times more sensitivity than H1 NMR spectroscopy.

16. Liquid chromatography combined with the NMR spectroscopy (LCNMR) can be used for simplifying the biofluids' complexity such as urine and fecal water extracts, that are not detected and quantified via 1D or 2D NMR. Similarly, LC-NMR-MS is used for the structural elucidation and detection of novel metabolites

17. Fluoxetine drug metabolized to norfluoxetine via dealkylation reaction, to ensure the drug metabolism NMR spectroscopy can be used for detection or quantification of the drug metabolites without further confirmation tests. Irbesartan drug after metabolic reaction converts into hydroxyl irbesartan, which can be confirmed by NMR spectroscopy. Metabolism of pyragallol drug which is metabolized via sulfation reaction and converted into the dihydroxyphenyl-2-O-sulfate metabolite. For the metabolism of the benzoic acid, an amino acid conjugation reaction takes place and transformed the drug into hippuric acid, this metabolic process can be confirmed via NMR.

18. 2. GC-MS based metabolite identification and quantification In GC-MS the mass spectrometer is coupled with the gas chromatography. GC with great resolution separated the volatile compounds but cannot appropriately identify them. In MS, ions moved through the vacuum, and by their mass to charge ratio ionized atoms or molecules are separated. GC-MS spectroscopy are preferable for detection and quantification of the small molecular weight metabolites (<650 daltons ) such as small acids, hydroxyl acids, alcohols, sugars, amino acids, fatty acids, hormones, sterols, drugs, toxins, catecholamine, aromatic and several intermediate products of primary metabolism

19. In all GC–MS based metabolomics studies, capillary columns are used because of their great efficiency. GC-MS efficiency, selectivity and analysis time increase directly with the length of the column. Longer columns are used for complex samples and require more time for analyses vice versa. In untargeted GC–MS based metabolomics studies, generally 30 m columns and non-polar stationary phases (5% phenylarylene 95% dimethyl polysiloxane or 5% diphenyl 95% dimethylpolysiloxane) are used.

20. Assche et al. used GC–MS/MS in scan mode for the investigation of metabolomics profiles of transgenic Caenorhabditis elegans and have identified a total of 76 unique metabolites and 8 out of them were considerably different. In 2016, Karimpour et al. have studied the postprandial effect on human plasma metabolome by using the GC–TOF–MS technique and identified 200 unique mass features in the sample. Lu et.al identified 2482 mass features in human serum by using the GC– qTOF –MS.

21. 3. LC-MS/MS-based metabolite identification and quantification LC coupled with MS is composed of three main parts, including the ion source which converts molecules into ions, the mass analyzer which separates these ions on bases of m/z ratio and passed them towards detector and the final element of LC/MS is the detector which detects or identifies the separated components. Hypothetical spectra of unknown ions can be predicted through in silico splitting techniques. For the estimation of given molecule’s fragment ions, two main methods are employed. First is the rule-based predictor which can be applied to the fragmentation mechanisms obtained from the literature. The second one is the in silico fragmentation such as Fragment Identification

22. Computational techniques can provide great help in the identification of metabolites thus they are not much efficient to replace the experimental validation process employed for the identification of these metabolites. For nonpolar and weakly polar compounds, reverse phase liquid chromatography with C18 columns is used which provides high resolution. For the separation of polar, ionic and hydrophilic molecules, normal phase (NP) liquid chromatography can be used.

23. Hydrophilic interaction liquid chromatography (HILIC) is a novel mode of separation having high compatibility with mass spectrometer and improved resolution for the detection of polar analytes. HILIC combines the mobile phase used in reverse phase separation and the stationary phase used on NP mode.

24. Drug Metabolic enzyme Metabolites Biofluid Analytical method Omeprazole CYP2C19 S- hydroxyomeprazole and omeprazole sulfone plasma LC–MS/MS Amodiaquine CYP2C8 N -desethylamodiaquine Plasma and urine HILIC– MS/MS Buspirone CYP3A4 1-[2-pyrimidyl]-piperazine plasma LC–MS/MS Tolbutamide CYP2C9 5-hydroxysulfadiazine Plasma and urine LC–MS/MS Caffeine CYP1A2 Paraxanthine Plasma and urine LC–MS/MS Clarithromycin CYP3A4 and ABCB1 14-hydroxyclarithromycin plasma LC–MS–MS Tolbutamide Dehydrogen ase Carboxy tolbutamide Plasma and urine LC–MS/MS Debrisoquine CYP2D6 4-hydroxydebrisoquine Plasma and urine LC–MS/MS Midazolam CYP3A 1-hydroxymidazolam Plasma and urine LC–MS/MS

25. 4. Other suitable approaches for metabolite identification LC-MS instrumentation is critical for both screening, investigation, and structural interpretation. However, the non-MS approaches can also play a crucial role in such situations where just the MS data is not satisfactory. 1. Full scan Full mass scan technique permits the identification of almost all ionizable metabolites and provides comprehensive details regarding the molecular mass of drug metabolites because of its nonselective nature. Triple quadrupole mass analyzers have been used which drastically reduces the sensitivity. This problem may resolve by employing IT analyzers.

26. 2. Precursor ion and constant neutral loss scan The precursor ion and constant neutral loss scan mode are only used for tandem mass spectrometers and employed for the determination and identification of unknown drug metabolites. Numerous metabolites of phase II during the atomization, have loosed a definite group that can be used for precise scanning and isolation of these conjugates. 3. Multiple reaction monitoring The multiple reaction monitoring mode has more selectivity for the detection of the metabolite. In this approach, only a specific precursor ion or metabolite has allowed to pass through the first quadrupole which afterward has split in the collision cell and then the product ion separated from the second quadrupole.

27. 4. Single ion monitoring To surpass the low sensitivity problem of triple quadrupole mass spectrometer, the alternative is single ion monitoring. This offer low specificity and sensitivity as against multiple reaction monitoring, but it has been provided many advantages when the exact pattern of the analytes is not accurately forecasted. 5. Hydrogen/deuterium exchange LC-MS Hydrogen/deuterium exchange LC-MS can be employed to obtain information related to drug metabolic pathways, MS fragment product ion formation and for studying and differentiation of the structures of the isomeric metabolites.

28. 6. High resolution mass spectrometry For the identification and determination of metabolites, the more accurate analyzer in mass prediction is TOF instrument. It has been declared that by using the accurate mass TOF, various drugs can be detected 5-25 times better as compared to nominal mass TOF. 7. Product ion scan Product scan mode is employed for the structural determination and elucidation of scanned drug metabolites. In this mode, in the first quadrupole, a metabolite has been chosen which split in the collision cell and then the product ions are examined in the second quadrupole. To acquire more particular structural data, the multistage scan by utilizing ion trap instruments can be employed.

29. 8. Ion mobility time-of-flight mass spectrometry The Ion mobility time-of-flight mass spectrometry is suitable for segregating ions based on their mass-to-charge ratios. The major benefit of this technique is its ability to segregate the metabolite isomers which makes it a stronger bioanalytical technique for the analysis of complicated samples . 9. Chemical agents for metabolite identification Particular chemical entities are capable to interact with few kinds of the metabolites as a result of respective signal frequencies are altered in both MS and NMR spectra. This feature has been frequently employed for the determination and detection of metabolites. In this approach, two 15 N labeled agents, one is aminooxy probe and the second one is cholamine tag is used. These labels have high specificity and can covalently bound with metabolites having carbonyl and carboxyl groups correspondingly. This procedure permits direct visibility of the metabolite’s signals in 2D 15N-1H-HSQC-NMR spectra and mass spectra.

30. Conclusion Various enzymes catalyze the biotransformation of drugs and are mainly classified into four categories based on the reactions catalyzed by these enzymes. Targeted and untargeted metabolomics strategies are used for the identification, detection, and quantification of endogenous and exogenous metabolites. Metabolite quantification is very crucial when drug metabolite is toxic or pharmacologically active. Mainly three bioanalytical techniques have been used in metabolomics analysis, including NMR, GC-MS and LC/MS/MS. Currently, LC-MS based metabolomics analysis has been frequently used because of the several advantages over the other two techniques. LC/MS/MS computational framework for the untargeted metabolomics, significantly helpful in the identification of known and unknown metabolites.