Analysis of drugs in biological fluids

Question 1

Where is bioanalysis performed?

Answer 1

drug discovery
therapeutic durg monitoring
bioequivalence studies
forensic science
toxicology studies
dope testing

Question 2

Which is best for evaluating Cumulative toxicity?

Answer 2

hair and nail

Question 3

silica particles, thrombin

clot activator

Answer 3

may be added to vacuntainer tubes for rapid serum separation

Question 4

Anticogualants

Answer 4

Lavender-Top EDTA Tube

Question 5

Basic and lipophilic drugs bind to

Answer 5

gamma globulin- which is acidic

Question 6

Acidic and lipophobic drugs bind to

Answer 6

albumin.

Question 7

matrix

Answer 7

refers all to the compoenets in a sample besides the analyte.

Question 8

How to minimize matrix effect?

Answer 8

selective extraction procedure
change buffer, strenght and pH
use of internal standards
different ionzation process

Question 9

what are the steps of a bioanalytical method?

Answer 9

sampling
sample preparation/ extraction
separation
detection
validation
quantification.

Question 10

Types of sampling preparation or extraction

Answer 10

1. conventional techniques
2. novel method: micro-extraction techniques

Question 11

Examples of conventional methods in sample prepation/ extraction

Answer 11

protein precipitation
liquid- liquid extraction

Question 12

Which drugs degrade when exposed to high temperatures?

Answer 12

Simvastatin, aspirin, testosterone, and benzodiazepines (BZD) are temperature-sensitive.

💡 Tip: “SAT BZD” (Simvastatin, Aspirin, Testosterone, BZD) – imagine SAT scores melting under heat!

Question 12

Which drugs are sensitive to light degradation?

Answer 12

Vitamin D, Nifedipine, and Nisoldipine degrade when exposed to light.

💡 Tip: Think of “D for Degradation” and remember that Vitamin D is linked to sunlight, making it susceptible to light effects.

Question 13

Which drug is particularly sensitive to oxidation?

Answer 13

Levodopa.

Question 14

What is the purpose of protein precipitation (PP)?

Answer 14

It removes proteins from a sample by adding a precipitating agent, making it easier to analyze the drug/metabolite.

💡 Tip: PP = Protein Purging!

Question 15

Why is protein precipitation (PP) commonly used in drug discovery?

Answer 15

Simple & straightforward
Easily automated in lab workstations
Works well for less complex biological matrices (e.g., hepatocytes, microsomes)
Can be used for whole blood and urine samples

Tip: S.A.M.P. → Simple, Automated, Matrices, Plasma/Blood/Urine

Question 16

What is the key mechanism of protein precipitation (PP)?

Answer 16

PP disrupts protein solubility and causes denaturation/precipitation, making it easier to remove proteins from the sample.

💡 Tip: PP = Protein Purge through Denaturation!

Question 17

How do acids aid in protein precipitation?

Answer 17

They lower the pH, which disrupts protein structure and leads to precipitation.
Examples: Perchloric acid, Trichloroacetic acid, Formic acid

Question 18

: How do salts facilitate protein precipitation?

Answer 18

They cause dehydration and aggregation of proteins, leading to their removal.
Examples: Ammonium sulfate, Sodium tungstate

Question 19

What are the three main categories of precipitating agents in PP?

Answer 19

Organic solvents (Acetonitrile, Methanol)
Acids (Perchloric, Trichloroacetic, Formic acid)
Salts (Ammonium sulfate, Sodium tungstate)

💡 Tip: OAS → Organic, Acid, Salt!

Question 20

What Happens After Protein Precipitation?

Answer 20

Centrifugation is performed to separate proteins from the liquid supernatant.

Question 21

What can be done with the supernatant liquid after centrifugation?

Answer 21

Direct injection into the analytical instrument.
Dilution to adjust concentration.
Filtration to remove residual particles.
Additional centrifugation to further purify the sample.

💡 Tip: “DIFC” → Dilute, Inject, Filter, Centrifuge!

Question 22

What is a limitation of protein precipitation?

Answer 22

While proteins are removed, other endogenous compounds (e.g., plasma/serum components) remain and can interfere with mass spectrometry (MS) analysis due to the matrix effect.

💡 Tip: PP ≠ Perfect Purification!

Question 23

Where is the biological sample collected for PP?

Answer 23

In an EDTA bottle to prevent clotting.

💡 Tip: EDTA = “Easy Drug Testing Anticoagulant”!

Question 24

What is the purpose of vortex mixing?

Answer 24

It ensures thorough mixing of the sample with EDTA and precipitating agents. ## Footnote 💡 Tip: Vortex = "Violent Shaking" for Uniform Mixing!

Question 25

What is the speed range for homogenizing the sample?

Answer 25

70–100 rpm for a suitable time.

Question 26

How is the solvent dried after extraction?

Answer 26

Using a centrifuge with a nitrogen stream.

Question 27

Which precipitants have the highest efficiency (~99.8-99.9%)?

Answer 27

✅ Trichloroacetic acid (CCl₃COOH, 10%) ✅ Perchloric acid (HClO₄, 6%) ✅ CuSO₄–Na₂WO₄ & ZnSO₄–NaOH ## Footnote 💡 Tip: "CCl₃, HClO₄ = Top Precipitants!"

Question 28

What are the major steps in protein precipitation?

Answer 28

Add precipitant (e.g., acetonitrile). Shake & mix thoroughly. Centrifuge to separate proteins. Collect the supernatant for analysis. ## Footnote 💡 Tip: "Add, Shake, Spin, Collect!"

Question 29

What is the principle of protein precipitation (PP)?

Answer 29

A precipitant reduces protein solubility, causing proteins to aggregate and separate from the liquid phase. ## Footnote 💡 Tip: Less Solubility = More Precipitation!

Question 30

How efficient is acetonitrile in PP?

Answer 30

97.2% at 1.0 volume 99.7% at 2.0 volume ## Footnote 💡 Tip: Higher volume = Better precipitation!

Question 31

What is a major limitation of PP?

Answer 31

It removes proteins but not other endogenous compounds, leading to the matrix effect in mass spectrometry (MS).

Question 32

What is the basic principle of mass spectrometry (MS)? | 💡 Tip: "MS = Mass-to-Charge Analysis!"

Answer 32

Mass spectrometry measures the mass-to-charge ratio (m/z) of ions to identify and quantify molecules in a sample. ## Footnote 💡 Tip: "MS = Mass-to-Charge Analysis!"

Question 33

What are the three main components of a mass spectrometer?

Answer 33

Ion Source – Converts molecules into ions. Mass Analyzer – Separates ions based on m/z ratio. Detector – Measures ion intensity and generates spectra. ## Footnote 💡 Tip: "Ionization → Separation → Detection!"

Question 34

Name three common ionization methods in MS.

Answer 34

Electrospray Ionization (ESI) – Used for large biomolecules (e.g., proteins). Matrix-Assisted Laser Desorption/Ionization (MALDI) – Soft ionization for polymers & biomolecules. Electron Ionization (EI) – Common in GC-MS for small molecules. ## Footnote 💡 Tip: "ESI = Proteins, MALDI = Biomolecules, EI = Small Molecules!"

Question 35

Q: Name four types of mass analyzers and their functions.

Answer 35

Quadrupole – Filters ions by applying electric fields. Time-of-Flight (TOF) – Measures time taken by ions to reach detector. Ion Trap – Traps ions and ejects them sequentially. Orbitrap – Provides high-resolution mass accuracy. ## Footnote 💡 Tip: "Q-TIO: Quad, TOF, Ion Trap, Orbitrap!"

Question 36

Name five drug classes extracted via PP.

Answer 36

✔ Cardiovascular drugs – Propranolol, Amiodarone. ✔ Antipsychotics – Olanzapine, Fluoxetine. ✔ Steroids & Hormones – Testosterone. ✔ Opioids & Pain Relievers – Morphine, Codeine. ✔ NSAIDs – Diclofenac. ## Footnote 💡 Tip: "PP for CVS, Psych, Pain, Hormones!"

Question 37

What is the main purpose of LLE?

Answer 37

LLE separates compounds based on their solubility in two immiscible liquids (polar vs. non-polar). ## Footnote 💡 Tip: "LLE = Solubility-Based Separation!"

Question 39

What makes LLE superior to Protein Precipitation (PP)?

Answer 39

✔ Better sample clean-up – Removes more interferences. ✔ Less matrix effect – Reduces ion suppression in mass spectrometry. ✔ More flexibility – Works with various solvents & samples. ## Footnote 💡 Tip: "LLE = Cleaner & More Precise!"

Question 39

Name four organic solvents used in LLE.

Answer 39

✔ Ethyl acetate ✔ Dichloromethane ✔ Chloroform ✔ Hexane ## Footnote 💡 Tip: "EDCH: Ethyl-Di-Chlor-Hex!"

Question 39

What are the alternative names for LLE?

Answer 39

✔ Solvent Extraction ✔ Partitioning ## Footnote 💡 Tip: "LLE = Solvent Partitioning!"

Question 40

What are the four main steps of SPE? ## Footnote 💡 Tip: "CELE: Condition, Equilibrate, Load, Elute!"

Answer 40

✔ Conditioning – Activates the sorbent for interaction. ✔ Equilibration – Removes residual solvent & preps the sorbent. ✔ Loading – Sample is added, and analytes bind to sorbent. ✔ Washing & Elution – Contaminants are removed, and analytes are collected

Question 42

Why is conditioning important? ## Footnote 💡 Tip: "Conditioning = Prepping the sorbent!"

Answer 42

✔ Removes trapped air. ✔ Activates ligands on the sorbent surface. ✔ Improves sample flow & analyte interaction.

Question 43

What happens when the sample is loaded onto the sorbent? ## Footnote 💡 Tip: "Loading = Selective binding!"

Answer 43

✔ The target analyte binds to the sorbent. ✔ Contaminants do not bind and are washed away.

Question 44

Why is sample processing important for quantification? | 💡 Tip: "Clean sample = Reliable quantification!"

Answer 44

✔ Ensures accurate measurement of analytes. ✔ Removes interferences from biological matrices. ✔ Prepares sample for chromatographic or mass spectrometry analysis.

Question 45

What is the role of the internal standard (ISTD) in sample quantification? ## Footnote 💡 Tip: "ISTD normalizes the response for better accuracy!"

Answer 45

✔ Compensates for variations in sample preparation. ✔ Improves precision and accuracy in quantification. ✔ Added at a fixed concentration (e.g., 150 ng/mL).

Question 46

Why is Methyl Tertiary Butyl Ether (MTBE) used in sample processing?

Answer 46

✔ Extracts non-polar analytes from plasma. ✔ Helps in phase separation for clean supernatant collection. ## Footnote 💡 Tip: "MTBE = Effective solvent for liquid-liquid extraction!"