Identification & Quantification of Antiretroviral Drugs using HPLC‑MS
Identification and quantification of antiretroviral drugs in an unknown sample using HPLC‑MS.
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At a glance
- Outcome: Identified ARVs in an unknown sample + quantified those above LOQ
- Tools: Agilent 1100 HPLC + Thermo LTQ Orbitrap XL (ESI+)
- Skills: calibration models, residual/error analysis, LOQ/LOD reasoning
- Result highlight: two analytes were quantifiable, but concentrations suggested a likely prep/dilution issue rather than method failure
Overview
Can HPLC‑MS reliably identify and quantify antiretroviral drugs (ARVs) in a simulated biological matrix? I built calibration curves (1–100 µg mL⁻¹), tested an “unknown” sample (MK‑U), then assessed performance using residuals and quantification limits.
Objectives
- Prepare calibration standards for five ARV drugs
- Generate linear calibration curves (1–100 µg mL⁻¹)
- Analyse an unknown sample (MK‑U)
- Identify analytes using retention time
- Quantify detected drugs using regression models
- Evaluate method performance (LOD/LOQ, residuals, systematic error)
Scope
ARVs included:
Efavirenz Emtricitabine Lopinavir Ritonavir Tenofovir disoproxil fumarate (TDF)
Approach
- Prepared standards by serial dilution
- Ran samples on HPLC‑MS (ESI+, Orbitrap)
- Used adjusted peak area vs concentration to build linear models
- Checked residuals for bias and assessed LOQ/LOD suitability
Results
1) Calibration performance
Overall: strong linearity across all ARVs.
- R² range: 0.954–0.996
- Residuals: slight systematic error visible
- Low end (1 µg mL⁻¹): high relative error (consistent with LLOQ limitations)
Takeaway: calibration was good enough for identification and quantification within range, but lowest points carried larger uncertainty.
2) Unknown sample (MK‑U): what was detected?
Detected: Lopinavir Ritonavir Emtricitabine TDF
Not detected: Efavirenz
3) Quantification (above LOQ)
Only Lopinavir and Ritonavir exceeded LOQ thresholds:
| Analyte | Concentration (µg mL⁻¹) | Note |
|---|---|---|
| Lopinavir | 143.7 | above calibration range |
| Ritonavir | 310.6 | above calibration range |
Interpretation: both values were outside the validated range (1–100 µg mL⁻¹), strongly suggesting a dilution/preparation issue for MK‑U rather than instrument/method failure.
Interpretation
What worked well
- High sensitivity and clear response across the calibration range
- Reliable identification of ARVs in a simulated biological matrix
- Residual analysis provided useful diagnostics (bias vs random error)
What likely went wrong
- MK‑U concentrations were implausibly high and beyond the calibration range → most consistent with prep/dilution error
Improvements
- Replicate injections to reduce uncertainty
- Internal standards to improve quantitative robustness
- Direct dilutions from stock solutions to reduce compounding error
What I learned
- Practical experience with HPLC‑MS instrumentation
- Building calibration curves + regression interpretation
- Distinguishing systematic vs random error using residuals
- Applying LOQ/LOD logic to real data
- Communicating results clearly for non-specialists