Chapter 34 — Quiz

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Chapter 34 — Quiz

Twenty-two questions. The answer key follows, collapsed. Work through the whole set before opening it.

1. A mass spectrometer measures:

(a) The mass of a molecule directly
(b) The mass-to-charge ratio of ions
(c) The number of amino acids in a peptide
(d) The concentration of a compound in solution

2. Tandem mass spectrometry (MS/MS) adds which capability over conventional MS?

(a) It quantifies how much of a compound is present
(b) It detects D-amino acid substitutions
(c) It establishes sequence by measuring the masses of fragments
(d) It measures endotoxin

3. Which of the following errors would mass spectrometry be structurally unable to detect?

(a) A completely different peptide of much smaller mass
(b) A missing residue
(c) Substitution of a D-amino acid for the intended L-form
(d) An entirely non-peptide contaminant of distinct mass

4. HPLC purity is conventionally reported as:

(a) The mass of target peptide divided by the total mass of the sample
(b) The target peak's area as a percentage of total peak area
(c) The height of the target peak relative to baseline noise
(d) The number of peaks detected

5. Co-elution refers to:

(a) Two samples run on the same instrument
(b) Two species emerging from the column at the same time and appearing as one peak
(c) The use of two detectors simultaneously
(d) A peptide eluting at its predicted retention time

6. A peptide purity method monitored at 280 nm will be least able to detect:

(a) A peptide impurity containing tryptophan
(b) A peptide impurity containing tyrosine
(c) A residual inorganic salt
(d) A truncated form containing phenylalanine

7. Which is the characteristic impurity of solid-phase peptide synthesis, and also the one area-percent HPLC is least able to resolve?

(a) Heavy metals
(b) Deletion sequences differing by one residue
(c) Endotoxin
(d) Residual water

8. "Peptide content" (also called assay or potency) asks:

(a) Whether the molecule is the right one
(b) What fraction of detected material is the target
(c) How much target peptide is present by mass
(d) Whether the material is sterile

9. A vial can report 98% HPLC purity and still contain far less peptide by mass than its label states, principally because of:

(a) Instrument calibration error
(b) Counterion, residual water, and salts
(c) Deliberate adulteration
(d) Degradation during analysis

10. The counterion most commonly accompanying a synthetic peptide after reversed-phase purification is:

(a) Chloride
(b) Sulfate
(c) Trifluoroacetate
(d) Phosphate

11. A sterility test asks:

(a) Whether bacterial cell-wall material is present
(b) Whether viable organisms are present
(c) Whether the container closure is intact
(d) Whether the product will cause fever

12. Endotoxin is:

(a) A toxin secreted by living bacteria during growth
(b) Lipopolysaccharide from the outer membrane of Gram-negative bacteria
(c) A degradation product of peptides
(d) A residual synthesis solvent

13. Which statement about endotoxin is correct?

(a) It is destroyed by ordinary autoclave cycles
(b) It is removed by sterilizing filtration
(c) It survives both heat sterilization and sterile filtration
(d) A passing sterility test implies acceptable endotoxin levels

14. Chapter 19's formulation of the distinction is:

(a) Sterility asks whether anything is alive in the vial; endotoxin asks whether anything ever was
(b) Sterility is a process; endotoxin is a product
(c) Sterility is measured by culture; endotoxin is measured by mass
(d) Sterility applies to injectables; endotoxin applies to orals

15. Amino acid analysis establishes:

(a) Sequence
(b) Composition
(c) Stereochemistry
(d) Sterility

16. Edman degradation cannot be used on a peptide that:

(a) Contains proline
(b) Has a chemically blocked N-terminus
(c) Exceeds 10 residues
(d) Contains disulfide bonds

17. Which of the following can a certificate of analysis establish?

(a) That the vial in your possession matches the document
(b) How the material was handled after testing
(c) The identity of the sample analyzed, by mass spectrometry
(d) Its own authenticity

18. The general principle about certificates stated in §34.7 is:

(a) A certificate is only as good as the laboratory that issued it
(b) A certificate is a claim about a sample, not a property of a vial
(c) A certificate is valid for the shelf life of the product
(d) A certificate must be signed to be meaningful

19. A "specification" on a certificate is:

(a) A description of the method used
(b) A predetermined acceptance criterion the material must meet
(c) The instrument's detection limit
(d) The name of the batch

20. This book declines to state a failure rate for unregulated peptide products because:

(a) The data are proprietary
(b) Samples are not randomly drawn, the market shifts, definitions vary, and negative results are underpublished
(c) The failure rate is close to zero
(d) Testing programs have not been conducted

21. You cannot test sterility into a product because:

(a) Sterility tests are unreliable
(b) The test is destructive, forcing sampling, and sampling has low power against low-level contamination
(c) Sterility tests take too long
(d) Regulators do not accept end-product testing

22. The distinction between a pharmacopeial monograph and GMP is best stated as:

(a) A monograph is voluntary; GMP is mandatory
(b) A monograph applies to peptides; GMP applies to small molecules
(c) A monograph defines the specification; GMP is how you know the process reliably produces material meeting it
(d) A monograph is issued by a manufacturer; GMP is issued by a regulator

Answer key

**1 — (b).** A mass spectrometer measures mass-to-charge ratio of ions. Mass is inferred from $m/z$ once the charge state is determined, which is why deconvolution exists (§34.2). **2 — (c).** MS/MS fragments the isolated ion and reads sequence from the mass differences between fragments. It does not quantify, and it is as blind to stereochemistry as conventional MS. **3 — (c).** A D-amino acid substitution changes the mass by exactly zero. Shape changes; mass does not. This is a structural blindness of the method, not a resolution limit (§34.2). **4 — (b).** Target peak area divided by total peak area. Note what this is a percentage *of*: a ratio among detected peaks, not a mass fraction of the sample (§34.3). **5 — (b).** Two species emerging together and recorded as one peak. It is the fundamental limitation of separation, which is why specificity must be demonstrated rather than assumed. **6 — (c).** At 280 nm the detector responds to aromatic side chains. An inorganic salt has no such chromophore and contributes nothing to either the numerator or the denominator — it is not counted as an impurity, it is not counted at all (§34.3). **7 — (b).** Deletion sequences. They arise from the standard failure mode of the standard synthesis method and differ from the target by one residue, so their retention behavior is nearly identical and their area may be counted inside the target peak (§32.3, §34.3). **8 — (c).** How much target peptide by mass. This is the question a person actually has, and the one a purity figure does not answer (§34.4). **9 — (b).** Counterion, water, and salts are real mass that a purity calculation never examines. Note that (c) is wrong as the *principal* reason — this outcome requires no dishonesty at all. **10 — (c).** Trifluoroacetate, from the trifluoroacetic acid used in cleavage and in reversed-phase mobile phases. Pharmaceutical processes exchange it for an acceptable counterion as a defined step. **11 — (b).** Viable organisms, assessed by attempting to culture them. **12 — (b).** Lipopolysaccharide from the Gram-negative outer membrane — a structural molecule, not an organism and not a secreted product, which is exactly why killing the organism does not remove it. **13 — (c).** It survives both. (d) is the specific error the chapter exists to correct. **14 — (a).** Sterility asks whether anything is alive in the vial; endotoxin asks whether anything ever was. **15 — (b).** Composition. Order requires Edman degradation or MS/MS. AAA's other major role is quantitative: it is a primary method for peptide content (§34.4, §34.6). **16 — (b).** Edman requires a free N-terminus. Blocking is a common deliberate modification in peptide drugs. **17 — (c).** Identity of the *sample analyzed*. Options (a), (b), and (d) are precisely the four things §34.7 says a certificate cannot establish. **18 — (b).** A certificate is a claim about a sample, not a property of a vial. Note that (a) is true but much weaker — it locates the problem in laboratory quality, when the deeper problem persists even with a flawless laboratory. **19 — (b).** A predetermined acceptance criterion. Results without specifications cannot fail; they can only be reported. **20 — (b).** All four reasons. The absence of a defensible number is not reassuring — it means exposure cannot be calculated, and treating an absent number as a low number is a common error. **21 — (b).** The test destroys what it tests, so only a sample can be tested, and a modest sample has low probability of encountering a small contaminated fraction. The test result can be correct while the inference is wrong. Sterility is produced by a validated process and confirmed, weakly, by sampling. **22 — (c).** A monograph defines the specification; GMP is how you know the process reliably produces material meeting it. A monograph is checked by testing a sample; GMP is verified by inspecting a system, and cannot be reconstructed after the fact.