A version of this article previously appeared in the February 2016 edition of Pharmaceutical Outsourcing

Mass spectrometry has developed rapidly to become an important analytical technique. However, application to the analysis of biological specimens was at first limited because the ionization processes could only be applied to compounds of low molecular weight, and the instrument interfaces did not permit easy introduction of biological samples into the mass spectrometer. This changed dramatically in the 1980s when electrospray ionization was introduced, enabling the ionization of larger bio-molecules, as well as straightforward coupling of the mass spectrometer (MS) to liquid chromatography (LC). The use of a tandem mass spectrometer (MS/MS) provided additional specificity and sensitivity and LC-MS/MS has become the mainstay analytical technique in pharmaceutical research.

LC-MS/MS is now used for the quantification of large and small molecule drugs, or biomarkers in biological fluids. The advantages it has over other techniques include high sensitivity and excellent specificity compared to conventional LC detectors or immunoassays, allowing routine analysis at nanogram to picogram levels, over a wide dynamic range.

Bioanalytical Techniques

In the field of bioanalysis, LC-MS/MS and immunoassays are the preferred analytical techniques, and with the increased interest in the development of large molecule drugs such as peptides and proteins, their complementary nature should ensure continued use, as well as the development of hybrid analytical methods which bring together the strengths of both technologies.

LC-MS/MS is used for bioanalysis at all stages of drug development (discovery, preclinical and clinical phases I to IV). Service providers use the technique to provide high-quality data, within tight timelines, that meet the stringent demands of the regulatory authorities. The high performance characteristics of LC-MS/ MS allow high throughput of samples and generate data that is precise and accurate.

The following case studies demonstrate the versatility of the technique.

  • Determination of Goserelin in Lithium- Heparinized Human Plasma

Goserelin is a synthetic hormone similar to the gonadotropin-releasing hormone (GnRH) synthesized within the hypothalamus gland in the brain. Its regular use decreases the production of estrogen and testosterone in the blood and allows for the treatment of a variety of medical problems, including prostate cancer in men, breast cancer in women, and endometriosis.

In this study, an LC-MS/MS method was validated to allow the determination of goserelin in human plasma in a range from 50 to 25,000 pg/mL, using a sample size of 300 μL.

Goserelin and leuprorelin (Figure 1) were used as internal analytical standards and the LC-MS/MS method used to quantify goserelin in human plasma samples was validated with a lower limit of quantification of 50 pg/ mL and an upper limit of quantification of 25,000 pg/mL. The between-run and withinrun imprecision (expressed as CV%) did not exceed 9.67%, while the absolute values of the inaccuracy (expressed as RE%) were all below 3.5%, demonstrating that the method is precise and accurate within the validated range (Table 1). The study also went on to show that use of hemolyzed and hyperlipidemic plasma as a source of the human plasma did not affect the imprecision and the inaccuracy of the results.

Molecular Structures of Goserelin and Leuprorelin

Article 1 TAB1

Representative chromatograms of a blank sample and a plasma sample spiked at the lower limit of quantification (LLOQ) are presented in Figures 2 and Figure 3, respectively. Thus, a selective method for the determination of goserelin in human plasma was successfully validated following the recommendations of the FDA’s Guidance on Bioanalytical Method Validation (2001 and 2013)1,2 and the EMA Guideline on bioanalytical method validation (2012).3 This method can be used to support clinical trials and is especially complementary to available selective methods for the assay of estradiol and testosterone.

Article 1 Fig2

Article 1 Fig3

  • Simultaneous Quantification of cGMP and cAMP in Human Plasma

Cyclic adenosine-3’,5’-monophosphate (cAMP) and cyclic guanosine-3’,5’-monophosphate (cGMP) (Figure 4) are nucleotides that serve as intracellular signaling molecules released by cells to trigger physiological changes within cells. They are referred to as secondary messengers, and control the activity of neurons in the central nervous system (CNS), regulating metabolic processes and electrical signaling.

Article 1 Fig4

Levels of cAMP and cGMP increase in response to neurotransmitters and are down-regulated by phosphodiesterase (PDE)-catalyzed hydrolysis. These two molecules are therefore useful biomarkers for evaluating the biological activity of phosphodiesterase inhibitor (PDEI) drugs, which block the degradation of these nucleotides.

Radioimmunoassay (RIA) and enzymeimmunoassay (EIA) kits are typically used to analyze cAMP and cGMP in human plasma. These techniques have excellent sensitivity, but there can be issues with specificity and matrix interferences, and the costs of the kits are relatively high.

The use of a validated LC-MS/MS method to quantify these analytes presents several advantages:

  • Simultaneous analysis of both nucleotides in a single assay.
  • Improved precision/accuracy and robustness through the use of stable isotope-labelled internal standards.
  • Improved specificity.
  • Less prone to matrix effects.

A study was performed with the objective of developing and fully validating an LC-MS/MS method for the analysis of cGMP and cAMP in human plasma, using Stable Isotope Labelled Internal Standards (SILIS) for both analytes. An Applied Biosystems API4000 triple quadrupole mass spectrometer coupled to a Shimadzu LC-20AD XR HPLC pump and autosampler was used for sample analysis (Figure 5).

Article 1 Fig5

The results highlighted in Table 2 show that validation was successful, and the performance data obtained during method validation indicate that this method can be used to support clinical studies.

Article 1 TAB2

Low-Volume LC-MS/MS Assays

The ‘three Rs’ are the guiding principles for the greater ethical use of animals in testing, and aim to promote: replacement - the preferred use of non-animal methods over animal methods whenever it is possible to achieve the same scientific aims; reduction - the use of methods that enable researchers to obtain comparable levels of information from fewer animals, or to obtain more information from the same number of animals; and refinement - the use of methods that alleviate or minimize potential pain, suffering or distress, and enhance animal welfare for the animals used.

By reducing the assay volumes of the methods used to analyze samples, the total volume collected from each animal is lowered, and consequently the total number of animals used for a study can also be minimized.

Article 1 Fig6

The ever-increasing sensitivity of LC-MS/MS systems has facilitated the implementation of methods with smaller assay volumes. However, there are concerns that the accuracy and precision of quantification may not be sufficient when using traditional pipettes to manipulate low volumes (≤ 10μL) of biological fluids.4 Capillary microsampling (CMS) has been widely adopted as a means of handling these low volumes, as it allows sampling of a low volume of blood in the animal lab and, subsequently, accurate and precise sampling of a fixed low volume of plasma in the bioanalytical lab.5 However, CMS is still a relatively young technique and there has been an expressed need to determine the impact of various parameters (e.g. adsorption, homogeneity and stability of the sample in the capillary) on the quality of the data obtained. Evaluating the performance of a CMS assay and subsequently comparing results obtained from the analysis of study samples using a CMS assay versus a traditional assay may help to alleviate these concerns.

A study was carried out to study the effect of reducing the assay volume of an existing LC-MS/MS method for the analysis of a single analyte in dog plasma. The method was subsequently adapted so that CMS could be used to sample dog plasma with an assay volume of 10μL.

The study undertook a number of stages in its process. Firstly, it compared the results obtained using air-displacement pipettes and capillaries for various assay volumes. Then the existing LC-MS/MS assay was adapted so that the assay volume could be reduced from 50μL to 5 or 10μL, before validation of the CMS method using an assay volume of 10μL was performed. Finally, the validated CMS method was used to analyze samples from a preclinical study, and compared to the results with those obtained previously using the existing method (assay volume 50μL / air-displacement pipettes).

The tests performed using a 5μL assay volume were highly imprecise, and so a number of modifications were subsequently made to the assay to address these issues (for method validation results see Table 3):

Article 1 TAB3

  • Capillaries were placed in microtubes (1.4mL, polypropylene) prior to processing.
  • The volume of acetonitrile used for precipitation was scaled down (100μL > 80μL) in line with the lower volume of the plasma sample.
  • The concentration of the internal standard working solution was lowered and the standard was incorporated into the acetonitrile solvent used for precipitation.
  • Three vortexing steps were required instead of one to optimize the precipitation step prior to centrifugation.
  • The volume of supernatant taken subsequently was reduced from 100 to 50μL and the volume of mobile phase added to the supernatant was also reduced from 200 to 100μL.
  • Tests on 5μL and 10μL capillaries showed a significant negative bias and poorer precision for the former. This was presumed to be due to ‘clogging’ of the narrower 5μL capillary when precipitating directly with acetonitrile. The 10μL capillary was therefore preferred.

A 14-day preliminary toxicity study in beagle dogs measured concentrations of X in dog plasma. For 34 samples reanalyzed, for which concentration data could be obtained, the difference between the ADP value and the CMS value compared to the mean of both values was within ±20% for all samples.
Thus, an existing LC-MS/MS assay for the analysis of a single analyte in dog plasma was successfully adapted so that the assay volume could be reduced from 50 to 10μl and capillary microsampling (CMS) could be used for sampling.

In Summary

The increasing presence of large-molecule drugs (peptides, proteins) in pharmaceutical company pipelines is predicted to rise. Immunoassays already provide an analytical platform for the quantification of large molecules and, as demonstrated above, the technology required to apply LC-MS/MS to protein analysis is more readily available. Bioanalytical service providers will need to meet the challenges presented by the analysis of large molecules by investing in both techniques.


Nicolas Fourrier
Director Biomarker and Biopharmaceutical Testing
SGS Life Sciences, Poitiers, France


1. ‘Guidance for Industry - Bioanalytical Method Validation’, U.S. Department of Health and Human Services - Food and Drug Administration, May 2001
2. 'Guidance for Industry - Bioanalytical Method Validation’, U.S. Department of Health and Human Services - Food and Drug Administration, Draft Guidance, September 2013
3. Guideline on bioanalytical method validation – European Medicines Agency - Committee for Medical Products for Human Use (CHPM) – EMEA/CHMP/EWP/192217/2009, February 2012, EMEA/CHMP/EWP/192217/2009
4. EBF: reflection on bioanalytical assay requirements used to support liquid microsampling, White et al., Bioanalysis (2014) 6(19), 2581–2586
5. Capillary microsampling in the regulatory environment: validation and use of bioanalytical capillary microsampling methods, Nilsson et al., Bioanalysis (2013) 5(6), 731–738


This article is based on three posters presented by SGS Life Science Services at EBF 2015 held in Barcelona, Spain from November 18-20, 2015. Goserelin analysis studies were performed by Sandrine Coppee, Sophie Minet, Coralie Quinet, Romuald Sable and Bernard Jeanbaptiste of SGS Life Science Services, Wavre, Belgium; cGMP and cAMP studies were performed by Nathalie Plaud, Geoffrey Nibaudeau, Nicolas Fourrier and Alain Renoux of SGS Life Science Services, Poitiers, France; and the capillary microsampling study was performed by Bernadette Dupraz and Peter Fordham, who are also based at SGS Life Science Services, Poitiers, France.