Liquid chromatography-mass spectrometry (LC/MS) is a powerful integrated chemical technique that allows simultaneous separation of components from a mixture and analysis of each component based on mass.
LC/MS has been applied in various areas of medicine, pharmaceutics, forensics, and biological research.
At the upcoming “Mass Spectrometry” meeting in Singapore and at “PITTCON” in Philadelphia, several researchers and analysts will present the latest improvements in LC/MS instrumentation, as well as applications of the technology to specific medical and biological research topics.
According to Khaled Mriziq, Ph.D., applications manager, MicroLC at Eksigent, part of AB Sciex, one major area of enhancement in LC/MS involves the use of microscale flow, in which the flow rates utilized in the analysis are within the range of 5–200 µL/min.
“Microscale flow LC presents several advantages over traditional LC, such as low solvent consumption, the use of only a small volume of a sample, the possibility of boosting the sensitivity of the assay—all of these achieved in short run times,” explains Dr. Mriziq. He also further discussed that these smaller-scale flow rates are actually appropriate for running analyses on tiny columns with a diameter range of 0.3 to 1 mm.
“Our research has focused on applying LC/MS in providing solutions for a range of applications to environmental, clinical, and pharmaceutical topics. With these reduced flow rates, using our microscale LC can resolve separations as effectively—or even more successfully—than traditional HPLC,” claims Dr. Mriziq.
“LC/MS is very commonly used in drug metabolism and pharmacokinetic studies (DMPK) of pharmaceuticals and proteomes. These specific application areas have complex samples that need to be detected and identified. Furthermore, these studies require high sensitivity and high-throughput techniques. LC/MS provides valuable information about the molecular weight, structure, identity, quantity, and purity of a sample.”
Using microscale flow LC, Dr. Mriziq further clarified that there was a need to maintain low delay volumes, thus requiring rapid gradients for the analysis. “We intend to generate high-throughput data without jeopardizing the sensitivity of chromatography. Although the flow rates of our assay were lower compared to traditional HPLC, our microscale flow LC only requires 5% of the total solvent utilized in traditional HPLC,” he explains.
One of the main applications of LC/MS is on the development of tests in monitoring specific stages in a particular disease entity. According to Erin Chambers, principal applications chemist at Waters, the LC/MS method was employed to measure amyloid beta peptides 1-38, 1-40, and 1-42, which were putative Alzheimer’s disease biomarkers in human cerebrospinal fluid (CSF).
“Historically, amyloid peptides have been quantified using ELISA methods, which suffer from a lack of standardization, cross reactivity, insufficient accuracy and precision, and long, labor-intensive sample preparation. In addition, it is unclear as to whether ELISA methods measure only free amyloid peptides,” says Chambers.
“In my work, a novel sample preparation technique, which eliminates both protein binding and aggregation (through denaturation) and nonspecific binding, was developed in conjunction with a high pH LC method using sub-2 mm particles. The MS method I describe relies on b or y ion fragments generated in positive ion electrospray, providing a significant improvement in specificity over earlier published methods that used water loss fragments in negative ion mode.
“The use of more specific b and y ion fragments coupled to choosing multiple reaction monitoring (MRM) pairs at higher m/z ranges facilitated the successful development of the MS portion of our method. In addition, quadrupole mass range proved invaluable in allowing the use of these higher m/z pairs.”
Chambers also reported that her team’s method produced results that were accurate in terms of percentage points, allowing highly accurate and precise measurements of amyloid peptides.
“The use of high m/z MRM pairs ensures that only the species of interest were being measured, and this facilitated the achievement of detection limits in the 50 pg/mL range for the three amyloid peptides we were monitoring. Overspiked quality control samples prepared from human CSF were accurately measured to within several percentage points—even at the 40 pg/mL level.
“Another critical attribute of our work was the development and testing of an appropriate surrogate matrix for human CSF. Standard curves prepared in artificial CSF doped with a carrier protein were shown to be equivalent to human CSF curves for quantitation of human CSF samples.”
She also says that the high accuracy and precision of the method should facilitate the differentiation of diseased and normal patient populations and offer correlations between the putative biomarker with disease progression.