A systematic approach to development of analytical scale and microflow-based liquid chromatography coupled to mass spectrometry metabolomics methods to support drug discovery and development

Abstract

As the reliance on metabolic biomarkers within drug discovery and development increases, there is also an increased demand for global metabolomics methods to provide broad metabolome coverage and sensitivity towards differences in metabolite expression and reproducibility. A systematic approach is necessary for the development, and evaluation, of metabolomics methods using either conventional techniques or when establishing new methods that allow for additional gains in sensitivity and a reduction in requirements for amounts of a biological sample, such as those seen with methods based on microseparations. We developed a novel standard mixture and used a systematic approach for the development and optimization of optimal, ion-pair free, liquid chromatography-mass spectrometry (LC-MS) global profiling methods. These methods were scaled-down to microflow-based LC separations and compared with analytical flow ion-pairing reagent containing methods. Average peak volume improvements of 7- and 22-fold were observed in the positive and negative ionization mode microflow methods as compared to the ion-pairing reagent analytical flow methods, respectively. The linear range of the newly developed microflow methods showed up to a 10-fold increase in the lower limit of detection in the negative ionization mode. The developed microflow LC-MS methods were further evaluated using wild-type mouse plasma where up to a 9-fold increase in peak volume was observed.

Keywords: Increased sensitivity in metabolomics; LC-MS; Metabolic profiling; Metabolomics; Microbore columns; Microflow LC.

Figure 1.

Extracted ion chromatograms of all 62 analytes (10 ppm) present in a 500 ng/mL standard mixture analyzed using the conditions selected for further method optimization. A) Positive ESI mode detection - Top to bottom: column-mobile phase conditions: Waters HSS T3- FA, pH 2; Waters HSS SB C18- FA, pH 2; Phenomenex Luna Omega Polar C18- FA, pH 2; Imtakt Scherzo SS-C18-Mixed Mode. B) Negative ESI mode detection- Top to bottom: column-mobile phase conditions: Imtakt Scherzo SS-C18-AmAc, pH 5; Imtakt Scherzo SM-C18-AmAc, pH 5; Imtakt Scherzo SS-C18-Mixed Mode.

Figure 2.

Effect of mobile phase B composition on the peak area of 39 standard compounds in the positive ESI mode. The standard mixture (100 ng/mL) was separated on an Imtakt Scherzo SS-C18 column with a column temperature of 40 °C and the 20 min LC method. Mobile phase A was 0.1 % formic acid in water for all conditions tested. The injection volume was 10 μL. Data points and error bars represent average peak area results and standard deviation for three replicate analyses. The scale of the X-axis is log 10.

Figure 3.

Effect of mobile phase A composition on the peak areas of 49 standard compounds in the negative ESI mode. The standard mixture (100 ng/mL) was separated on an Imtakt Scherzo SM-C18 column with a column temperature of 40 °C and the 26 min LC method. Mobile phase B was acetonitrile for all conditions tested. The injection volume was 10 μL. Data points and error bars represent average peak area results and standard deviation for three replicate analyses. The scale of the X-axis is log 10.

Figure 4.

Comparison of the average peak volume from methods chosen for scale down to microflow-based LC separations in the A) positive ESI mode and B) negative ESI mode. The standard mixture (100 ng/mL) was separated on 0.3 × 100 mm columns with a column temperature of 40 °C using a 30 min LC method. The injection volume was 1 μL. Data points and error bars represent average peak volume results and standard deviation for three replicate analyses. The scale of the X-axis is log 10. FA: formic acid mobile phases; AmAc: ammonium acetate mobile phases; Mixed: mixed-mode methods; AmF: Ammonium formate; NH₄F: Ammonium fluoride

Figure 5.

Comparison of analyte peak volumes for the standard mixture (100 ng/mL) analyzed using the optimized microflow, RP analytical flow, and ion-pair reagent containing methods. A) Positive ESI mode results. B) Negative ESI mode results. The injection volume was 0.8 μL for all methods. Data points and error bars represent average peak volume results and standard deviation for three replicate analyses. The scale of the X-axis is log 10.

Figure 6.

Comparison of a 50x diluted WT mouse plasma sample analyzed using the optimized microflow, RP analytical flow, and ion-pair reagent containing methods. A) Positive ESI mode results. B) Negative ESI mode results. The injection volume was 0.8 μL for all methods. Data points and error bars represent average peak volume results and standard deviation for three replicate analyses. The scale of the X-axis is log 10.