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.
LC/MS has also been extensively used in the research work conducted by Jaran Jainhuknan, Ph.D., Southeast Asia applications support manager at Bruker Daltonics. “In our study, we wanted to identify changes in the metabolism of two arginine yeast biosynthesis pathway deletion mutants. Since polar metabolites of the arginine biosynthetic pathways are poorly retained in reversed phase (RP) C18 chromatography, we derivatized them by dansylation, according to a previously published protocol of an independent group.
“This rendered the target compounds, which contained primary or secondary amine or phenolic functional groups, less polar and led to increased retention in RP-LC. Coupling this RP-LC to a high-resolution quadrupole time-of-flight (QTOF) instrument, in our case a Bruker maXis Impact, enabled us to detect and identify the compounds of interest by making use of the high mass accuracy obtained in MS and MS/MS spectra,” discusses Dr. Jainhuknan.
He also explained that the application of a nontargeted profiling technique facilitated the determination of metabolic pathway perturbations in the deletion mutant yeast strains based on the established derivatization protocol followed by subsequent RP-LC QTOF LC/MS analysis. “This strategy has revealed an accumulation of precursor metabolites that occur prior to the blockage of enzymatic reactions,” he says.
LC/MS technology has also been applied to the separation and analysis of plant natural products and metabolites. “Most bioactive compounds from plants are medically important, yet their extraction is often challenging due to the occurrence of isobars and isomers,” said Daniel Cuthbertson, Ph.D., applications scientist, metabolomics at Agilent Technologies.
“Using LC/MS, we have detected more than 500 bioactive compounds from plants, and through collaborations with the academe, we have established a database of known plant natural products. Aside from generating data on peaks and masses of different plant metabolites, this database also provides information on the best conditions on how to isolate each plant metabolite, elution methods on a column, retention time, as well as ratios of specific ions such as sodium and other protons.”
Dr. Cuthbertson noted that these LC/MS-derived molecular features have driven some of the compounds into clinical trial-level investigations as novel therapeutic regimens for particular diseases.
“Our next goal is to further use LC/MS in generating more focused libraries—possibly having a database that is more specific to a biological matrix. Once we have this in place, various classes of metabolites will then be easier to identify, such as taxanes and terpenes.
“In addition, the origin of a particular metabolite can also be quickly determined. For example, a marine natural product must have a specific signature that allows an analyst to immediately rule out that it comes from a plant. Using this type of approach, the chances of generating false-positive readouts during separation analysis would be significantly lower.”
Dr. Cuthbertson’s group also envisions conducting investigations on particular biochemical pathways involving their natural products.
Drug Abuse Testing
The sensitivity and precision of an LC/MS assay also highly relies on the quality and power of the instrument. “The Shimadzu ultra high performance liquid chromatography (UHPLC) LC-30A plays an important role in the rapid separation of components,” says Lin Cai-Yong of Shimadzu.
“When UHPLC is coupled with the MRM of tandem mass spectrometry (MS/MS), matrix effects of samples could be eliminated, thus resulting in a highly sensitive analysis of compounds, even at trace levels,” Cai-Yong explains.
His group has used LC/MS in establishing a rapid analytical technique in determining levels of specific drugs, including amphetamines, which are psychostimulants that have often been misused. “By using urine samples, we are capable of detecting the concentrations of commonly abused drugs using UPLC coupled with triple quadrupole mass spectrometry,” he adds.
“In order to develop a rapid, convenient, and sensitive analytical method for detection, we used the Shimadzu LCMS-8040 instrument to optimize our chromatography separation conditions, such as precursor and product ion collision electron voltages. Each material has two couple ions, that is to say, one is qualitative couple ion, whereas the other serves as a quantitative couple ion,” says Cai-Yong.
“The matrix effects of the target materials were thereafter calculated. If the matrix effect fell within the range of 80–120%, we can then establish the calibration using standard solutions through dilution.” Cai-Yong further explains that this methodology requires cautious monitoring.
“The limit of detection and the recovery of spikes of the identified drugs were examined and subsequently adopted for actual-sample analysis. Certainly, if the sample pretreatment method was complex, the influence of the extraction and desorption conditions should also be optimized to obtain high sensitivity,” he says.
The Right Containers
In terms of containers used in LC/MS assays, Dave Edwards, product manager of vials and closures at Thermo Fisher Scientific, explains that glass vials may introduce contaminants during analysis. “Previous glass vial products used in LC/MS tests were thought to be clean, although with more sensitive detectors, analysis eventually shows the presence of contaminants. You might thus be seeing things that might not be in your sample and instead, these were present in your glass vial,” Edwards points out.
“Thermo Fisher has manufactured LC/MS vials that have undergone lot-to-lot testing; each vial package of 100 comes with a certification that says this specific lot has been tested. Our glass vials are subjected to intense quality control,” he says.
“Our multistep procedure for cleaning glass vials significantly reduces the number of contamination cases in LC/MS assays that can be attributed to glass vials,” Edwards claims. “Our analysis is performed on each component—the septa, cap, and vial as a set.”
Edwards also explains that most developing countries may be at a disadvantage when performing LC/MS, with laboratories rewashing vials and caps for the next test or assay. He is concerned that these types of quality control issues may be harder to resolve when management drives a laboratory to find ways in minimizing the cost of lab items, including glass vials for LC/MS.
“It is important to use certified clean vials in your analysis. With constructive feedback from our clients, we have used their input in contributing to the improvement of specific practices in generating analytical data,” Edwards says.