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Sensitive Quantification of Drug Metabolites Using LC-MS

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Determining what happens to a drug after it enters the body is a key consideration in the drug development process. Without studying its’ metabolic fate, and understanding the resulting pharmacological responses, it is impossible to evaluate the efficacy and safety of a drug. But the detection and quantification of metabolites is not a simple process, especially due to the low abundance of metabolites in biological samples, and the complex nature of the matrices involved. In this respect, liquid chromatography coupled with mass spectrometry (LC-MS) has emerged as one of the most powerful analytical tools for the screening and identification of drug metabolites.1  Advances in the field of LC-MS have allowed scientists to analyze drug metabolic pathways and drug-drug interactions more closely, and improve therapeutic strategies for many challenging diseases.


Dr. Mohd Yusmaidie Aziz, a researcher at the Advanced Medical and Dental Institute in Universiti Sains Malaysia, has spent years studying metabolites of anti-malarial drugs, such as piperaquine, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. He explains that pharmaceutical scientists often prefer LC-MS/MS to other analytical techniques, when quantitating metabolites, because of its applicability and sensitivity.  

“A decade ago, high performance liquid chromatography (HPLC) was commonly used for drug quantitation,” says Aziz. “Compounds were identified based on UV, fluorescence, or electrochemical detection. But nowadays, pharmaceutical researchers have access to more advanced technologies such as mass spectroscopy with higher sensitivity, resolving power, and broad dynamic range. Especially when working with biological samples that are limited, sensitivity is an important factor.” 

According to Aziz, the LC-MS/MS methods he uses provide high enough selectivity and sensitivity to analyze the parent drug together with its metabolites. “During LC-MS/MS operation, a compound will be ionized and fragmented to specific product ions of the compound and that is considered as a fingerprint,” he says. “In addition, LC-MS/MS has a very high level of sensitivity to detect trace amounts of the compound.” 

The technique allows molecules to be detected at the parts per trillion (PPT) level, providing a means for Aziz’s team to measure the concentration of piperaquine and its metabolites – which are even lower than the parent drug concentration – in human plasma. 

Assaying Anti-Malarial Drug Metabolites

Piperaquine is an anti-malarial drug commonly used in combination with dihydroartemisinin because of its long biological half-life.2  When piperaquine enters the body, it is metabolized in the gut and the liver to compounds that have equal or longer half-lives, sometimes lasting 3-4 weeks.2  

“Our interest in quantifying piperaquine metabolites stemmed from the fact that none of these compounds had previously been assayed in systemic circulation,” explains Aziz. “Determining the concentrations of piperaquine in human plasma can help us to further study the effects of metabolites in systemic circulation and their potential for drug-drug interactions even after the treatment has ended.”

Using LC-MS/MS, Aziz and his collaborators at Sahlgrenska Academy, University of Gothenburg, Sweden, developed a sensitive and quantitative assay for piperaquine and two of its metabolites. The technique involved separating analytes from plasma samples on a C18 column and detecting them on a tandem mass spectrometer with an ESI source operated in the positive ion mode with deuterated piperaquine as an internal standard.3  Detection and quantitation was based on the mass-to-charge ratios (m/z) of precursor-product ion pairs, which are specific to piperaquine and its metabolites.

“Before running samples, we made sure to validate the method based on FDA guidelines for bioanalytical methods,” says Aziz. “There are criteria in the guidelines that needs to be strictly followed when establishing the method. Parameters include accuracy, precision, stability, recovery and matrix effects.”

Quantifying Metabolites of HIV Therapy

For people suffering from acquired human deficiency syndrome (AIDS) brought on by human immunodeficiency virus-type 1 (HIV-1), the recommended first line of therapy includes a combination of two nucleoside reverse transcriptase inhibitors (NRTIs).4  In essence, these molecules can stop the HIV from making copies of itself when it enters a healthy human cell. NRTIs go about this process by inhibiting an enzyme known as reverse transcriptase, but first, the molecules need to be metabolized to their active form.

“NRTIs are actually prodrugs,” says Dr. Yazen Alnouti, associate professor at the Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center (UNMC). “They require intracellular phosphorylation to active triphosphate (TP) nucleotide metabolites before they can inhibit the HIV reverse transcriptase.”

Monitoring these pharmacologically active metabolites inside cells is difficult due to their hydrophilicity, instability, and low concentrations in blood and tissues. Because hydrophilicity of TP metabolites makes separation using traditional reverse phase chromatography ineffective, alternative approaches are used and can be categorized as direct and indirect LC-MS methods.5  The direct approach relies on the direct quantification of the nucleotide metabolites under non-reverse phase liquid chromatography conditions. On the other hand, the indirect methods involve quantifying the parent nucleosides by dephosphorylation of the metabolites under reverse phase liquid chromatography conditions. Alnouti’s team investigated both direct and indirect approaches to quantify NRTIs lamivudine (3TC) and abacavir (ABC), and their metabolites.

“Most existing methods that measure TP metabolite concentrations in peripheral blood mononuclear cells (PBMCs) require sourcing these cells from large volumes of human blood (6-18 ml),” explains Alnouti. “We wanted to develop and validate a sensitive, selective LC-MS/MS method that can be used to quantify TP metabolites in PBMCs coming from much smaller blood volumes (~0.5 ml).” 

A Sensitive LC-MS/MS Method to Measure NRTI Metabolites

Researchers from Alnouti’s team at UNMC were able to simultaneously measure metabolites from two separate NRTI drugs in mouse blood. They also quantified these metabolites in mouse immune cells obtained from spleen, liver, and lymph nodes. The lead author of the study, Dr. Nagsen Gautam, now an assistant professor at UNMC, clarified that their technique involved isolating TP metabolites using anion exchange chromatography, converting them to the parent nucleosides via dephosphorylation, and analyzing these parent nucleosides as surrogates for their TP metabolites using a sensitive LC-MS/MS method.6 He further explained that for targeted quantification of small metabolite molecules, most triple-quadrupole LC-MS/MS systems in multiple reaction monitoring mode (MRM) will grant high sensitivity and selectivity. 

Their results showed that the lower limits of quantification were 10 pg/ml for the 3TC metabolite, and 4 pg/ml for ABC metabolite.6 According to Gautam, these results suggest that his team’s method is 12.5- to 50-fold more sensitive than the previous direct and indirect LC-MS/MS methods measuring 3TC and ABC triphosphate metabolites. “In addition to optimizing sensitivity, we also improved conditions to ensure the stability of TP samples and standards during sample collection, preparation, LC-MS/MS analysis, and storage. These factors are typically not addressed in other methods,” he added. The exact conditions can be found in Gautam’s recently published paper in the Journal of Pharmaceutical and Biomedical Analysis.6

Having an accurate method to monitor the levels of pharmacologically active metabolites in limited blood and tissues samples will help to speed up the preclinical and clinical development of more efficacious antiretroviral therapy for patients afflicted by HIV/AIDS.  Armed with their optimized LC-MS technique, Alnouti’s team is now focused on tracking TP metabolites in hidden viral reservoirs in the body. “HIV resistance and relapse after years takes place due to release of the virus from hidden viral reservoirs, mainly lymph nodes,” Alnouti explains. “Monitoring active intracellular TPs in these hidden reservoirs can support the optimization of new therapies that have the potential to attack the virus in these secret spots and completely eradicate the virus.”


1. Xiao, J. F.; Zhou, B.; Ressom, H. W., Metabolite identification and quantitation in LC-MS/MS-based metabolomics. TrAC Trends in Analytical Chemistry 2012, 32, 1-14.

2. Tarning, J.; Bergqvist, Y.; Day, N.P.; Bergquist, J.; Arvidsson, B.; White, N. J., Characterization of human urinary metabolites of the antimalarial piperaquine. Drug Metab. Dispos. 2006, 34 (12), 2011–2019.

3. Aziz, M. Y.; Hoffmann, K.-J.; Ashton, M., LC–MS/MS quantitation of antimalarial drug piperaquine and metabolites in human plasma. Journal of Chromatography B 2017, 1063, 253-258.

4. Panel on Antiretroviral Guidelines for Adults and Adolescents: Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. December 1, 2009; 1-161.

5. Jansen, R.S.; Rosing, H.; Schellens, J.H.; Beijnen, J.H. Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs. Mass Spectrom. Rev. 2011, 30 (2) 321–343.

6. Gautam, N.; Lin, Z.; Banoub, M. G.; Smith, N. A.; Maayah, A.; McMillan, J.; Gendelman, H. E.; Alnouti, Y., Simultaneous quantification of intracellular lamivudine and abacavir triphosphate metabolites by LC–MS/MS  Journal of Pharmaceutical and Biomedical Analysis 2018, 153, 248-259.