Phytochemical fingerprinting: HPTLC, LC-MS and GC-MS applications MCQs With Answer

Introduction: Phytochemical fingerprinting: HPTLC, LC-MS and GC-MS applications MCQs With Answer is designed for M.Pharm students to strengthen practical and theoretical understanding of chromatographic fingerprinting used in herbal drug analysis. This concise quiz-focused blog covers principles, instrumentation, sample preparation, ionization techniques, derivatization, data interpretation and chemometric integration with emphasis on HPTLC, LC-MS and GC-MS workflows. Questions target method development, validation, qualitative and quantitative profiling, metabolite identification, marker selection and regulatory relevance in quality control. The set challenges students to apply knowledge to real-world herbal standardization and research scenarios, preparing them for exams and laboratory decision-making.

Q1.
Which characteristic of HPTLC makes it particularly suitable for routine herbal fingerprinting in quality control laboratories?

• High-resolution mass analysis for unknown identification
• Ability to analyze multiple samples in parallel on a single plate
• Direct coupling to electrospray ionization for MS
• Requirement for volatile analytes only

Correct Answer: Ability to analyze multiple samples in parallel on a single plate

Q2.
In LC-MS profiling of plant extracts, which ionization technique is most commonly used for polar, thermolabile secondary metabolites such as glycosides and flavonoid glycosides?

• Electron ionization (EI)
• Atmospheric pressure chemical ionization (APCI)
• Matrix-assisted laser desorption ionization (MALDI)
• Electrospray ionization (ESI)

Correct Answer: Electrospray ionization (ESI)

Q3.
For GC-MS analysis of essential oils from a medicinal plant, which sample preparation step is most appropriate prior to injection?

• Direct dilution of aqueous extract without extraction
• Derivatization to form trimethylsilyl derivatives
• Steam distillation or headspace sampling to enrich volatiles
• Protein precipitation with acetonitrile

Correct Answer: Steam distillation or headspace sampling to enrich volatiles

Q4.
Which HPTLC detection mode provides enhanced specificity for phenolic compounds by visualizing as colored spots after reagent treatment?

• UV 254 nm illumination without reagent
• Fluorescence detection at 366 nm only
• Post-chromatographic derivatization with natural product–PEG reagent
• Direct scanning by refractive index detector

Correct Answer: Post-chromatographic derivatization with natural product–PEG reagent

Q5.
Which parameter is most critical in LC-MS method development for distinguishing isobaric plant metabolites?

• Chromatographic resolution and retention time separation
• Use of high injector temperatures
• Increasing sample injection volume indefinitely
• Switching to single quadrupole only

Correct Answer: Chromatographic resolution and retention time separation

Q6.
In GC-MS, electron ionization (EI) provides which advantage useful for phytochemical fingerprinting?

• Generation of extensive fragmentation patterns aiding library matching
• Soft ionization preserving molecular ion for large glycosides
• Compatibility only with polar nonvolatile compounds
• Direct quantitation without calibration curves

Correct Answer: Generation of extensive fragmentation patterns aiding library matching

Q7.
Which chemometric technique is commonly applied to HPTLC or LC-MS fingerprints for classifying plant samples by geographic origin?

• Principal component analysis (PCA)
• Fourier transform
• Simple linear regression without multivariate data
• Basic moving average smoothing

Correct Answer: Principal component analysis (PCA)

Q8.
What is the main rationale for using internal standards in quantitative LC-MS fingerprinting of herbal extracts?

• To increase chromatographic retention of analytes
• To correct for variability in extraction, ionization and instrument response
• To replace external calibration curves entirely
• To change the polarity of analytes

Correct Answer: To correct for variability in extraction, ionization and instrument response

Q9.
Which mass analyzer is preferred when high mass accuracy is required for elemental composition determination in metabolite identification?

• Quadrupole (single)
• Time-of-flight (TOF) or Orbitrap
• Ion trap without high resolution
• Flame ionization detector

Correct Answer: Time-of-flight (TOF) or Orbitrap

Q10.
During HPTLC method validation for fingerprinting, which parameter assesses repeatability of spot Rf and densitometric response under the same conditions?

• Intermediate precision
• Limit of detection (LOD)
• System suitability and precision (repeatability)
• Robustness to mobile phase composition

Correct Answer: System suitability and precision (repeatability)

Q11.
What is the principal limitation of GC-MS compared to LC-MS for analysis of complex plant extracts?

• GC-MS cannot provide any structural information
• GC-MS requires analytes to be volatile or derivatized, limiting polar nonvolatile metabolites
• GC-MS has unlimited mass range for high molecular weight polymers
• GC-MS always produces intact molecular ions for all compounds

Correct Answer: GC-MS requires analytes to be volatile or derivatized, limiting polar nonvolatile metabolites

Q12.
Which practice improves metabolite coverage in LC-MS based fingerprinting of herbal medicines?

• Using a single isocratic method only
• Employing multiple chromatographic modes (reverse-phase and HILIC) and ionization polarities
• Analyzing only the most abundant peaks and ignoring low-abundance ions
• Omitting sample cleanup entirely to preserve all components

Correct Answer: Employing multiple chromatographic modes (reverse-phase and HILIC) and ionization polarities

Q13.
In LC-MS/MS (tandem MS) targeted quantitation, what is the advantage of using multiple reaction monitoring (MRM)?

• It increases the number of possible adducts detected
• It provides high specificity and sensitivity by monitoring defined precursor→product ion transitions
• It eliminates the need for chromatographic separation
• It only works for GC-MS methods

Correct Answer: It provides high specificity and sensitivity by monitoring defined precursor→product ion transitions

Q14.
Which derivatization reagent is commonly used to increase volatility and detectability of sugars and amino acids for GC-MS profiling?

• Phenyl isothiocyanate (PITC) without volatile derivatives
• Trimethylsilylation reagents like BSTFA or TMCS
• EDC/NHS carbodiimide coupling
• Sodium borohydride reduction only

Correct Answer: Trimethylsilylation reagents like BSTFA or TMCS

Q15.
When constructing a phytochemical fingerprint for regulatory acceptance, which of the following is most important?

• Documented method, validated parameters, defined markers and acceptability criteria for batch comparison
• Only high-resolution MS data without retention or chromatographic details
• Using a single reference batch without system suitability checks
• Random visual inspection of chromatograms without numeric criteria

Correct Answer: Documented method, validated parameters, defined markers and acceptability criteria for batch comparison

Q16.
In LC-MS fingerprinting, what is the role of adduct formation (e.g., [M+Na]+ or [M+H]+) in data interpretation?

• Adducts provide no useful information and must be ignored
• Adduct patterns help infer molecular weight and ionization behavior, aiding identification
• Adducts convert all analytes into identical fragments
• Adduct formation prevents chromatographic separation

Correct Answer: Adduct patterns help infer molecular weight and ionization behavior, aiding identification

Q17.
Which approach is most effective for distinguishing structurally related isomers in LC-MS phytochemical profiling?

• Relying solely on nominal mass from single quadrupole
• Combining chromatographic separation, high-resolution MS and MS/MS fragmentation patterns
• Using only UV spectra without MS data
• Using derivatization that makes all isomers co-elute

Correct Answer: Combining chromatographic separation, high-resolution MS and MS/MS fragmentation patterns

Q18.
What is a practical advantage of using HPTLC coupled with densitometry for herbal standardization compared with LC-MS?

• HPTLC always provides exact molecular structures
• Lower instrument cost, visual comparison of many samples and simple semi-quantitation suited for routine QC
• HPTLC requires no method validation
• HPTLC eliminates need for sample extraction

Correct Answer: Lower instrument cost, visual comparison of many samples and simple semi-quantitation suited for routine QC

Q19.
Which factor is critical when selecting marker compounds for phytochemical fingerprinting intended for batch-to-batch consistency?

• Markers should be extremely rare trace compounds only
• Markers should be biologically irrelevant and highly variable
• Markers should be characteristic, reproducible, abundant enough for reliable detection and relevant to activity or identity
• Markers should be chosen arbitrarily without supporting data

Correct Answer: Markers should be characteristic, reproducible, abundant enough for reliable detection and relevant to activity or identity

Q20.
Which validation parameter specifically assesses the lowest concentration of an analyte that can be detected but not necessarily quantified in LC-MS fingerprinting?

• Limit of quantitation (LOQ)
• Linearity range
• Limit of detection (LOD)
• Accuracy at high concentration

Correct Answer: Limit of detection (LOD)

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