LC/MS analysis has long delivered a high standard for characterizing drugs and analyzing them throughout clinical development. Although significant gains have been made in the sensitivity of MS in recent years, conventional LC/MS workflows still lack the sensitivity required for the routine quantitation of challenging targets. Targets such as synthetic peptides and biologics are often very potent and are therefore available only at low concentrations in biological matrices; they require exceptionally sensitive analysis techniques to accurately quantitate and characterize them during development. One way to greatly improve the sensitivity of LC/MS is to lower the flow rate of analytical flow LC (>200 µL/min) by up to two orders of magnitude, to microflow rates (1–50 µL/min). This enables greater ionization efficiency, delivering a higher signal-to-noise ratio and a reduction of lower limits of quantitation (LLOQ). Additionally, utilization of microflow rates is cost-effective and greener chemistry: reducing the flow rate by two orders of magnitude subsequently reduces solvent use on a similar scale, drastically reducing waste and costs.
Lowering the flow rate is, however, not without challenges. The use of microflow LC requires the use of smaller i.d. tubing, which can clog more easily. Further, if leaks occur, they are more difficult to determine visually at low flow rates. Another barrier to the uptake of microflow LC is the time it takes to load samples at microflow rates. This slow sample loading increases overall run times considerably, and is an important consideration for busy research laboratories.
To address these challenges, a Trap-Elute MicroLC-MS method has been developed using the SCIEX M5 MicroLC (SCIEX, Concord, Ont., Canada). The system is simple to implement due to its use of fingertight fittings to the column; this, along with the system’s compatibility with any microflow column, allows challenging targets to be investigated. In addition, the system’s source requires no manual adjustments to optimize the spray, enhancing its ease-of-use. By loading the sample onto a trap column at fast speeds prior to elution at microflow speeds, the method provides the high sensitivity of microflow LC without incurring long and costly run times.
Improving analysis of challenging targets
Advances made in producing effective biologics and synthetic peptides have the promise to deliver important treatments for patients worldwide, yet challenges remain for researchers in the pharmaceutical industry to truly unleash their potential. Biologics and synthetic peptides are frequently very costly to produce, even in small quantities, and must be analyzed not only during production but also in complex biofluids throughout multiple stages of clinical development. These novel drugs require highly sensitive analytical techniques to become routine and accessible. The Trap-Elute MicroLC-MS system answers this need by enabling methods that use small sample sizes and short run times combined with accurate, sensitive quantitation.
In order to explore the versatility of the Trap-Elute MicroLC-MS system and compare it to conventional methods, the microflow LC workflow has been applied to difficult targets comprising both small and large molecules. The small-molecule target chosen for analysis was desmopressin, a 1-kDa synthetic analog of human vasopressin used to treat overproduction of urine and blood-clotting disorders. This method was also used to analyze a large biologic, the antibody-drug conjugate (ADC) trastuzumab emtansine used in the treatment of breast cancer. In both cases, the sensitivity of analysis has been greatly improved over analytical flow LC.
Trap-Elute microflow LC
The Trap-Elute microflow LC method has been developed and optimized to enhance sensitivity and reduce LLOQ values for challenging targets including both small and large molecules. The method utilizes sample-specific extraction procedures, followed by reconstitution and sample loading onto a trap column at high flow rates. This step is crucial to remove a major barrier for the general uptake of previous microflow methods—the long sample loading times. Once loaded onto the trap column, samples are eluted over a second column at microflow speeds prior to MS analysis. A detailed general method for Trap-Elute MicroLC-MS of desmopressin and trastuzumab emtanzine is described below.
Following extraction, samples were loaded onto a SCIEX M5 MicroLC in Trap-Elute mode for separation, followed by MS analysis using a SCIEX QTRAP 6500+ system. Loading onto the initial trap column enables high-volume sample loading without extensive run times, as the flow rate for this step was 40 µL/min. Elution over the second column was then performed at the much slower flow rate of 5 µL/min in order to achieve the sensitivity gains of microflow separation. Column chromatography details for the analyte trapping and analyte separation steps can be found in Tables 1 and 2, respectively.
MS analysis was performed on a SCIEX QTRAP 6500+ system with OptiFlow Turbo V source with a 25-µm SteadySpray electrode. Importantly, this MS setup required no physical adjustment of the probe or electrode positions for microflow analysis, significantly reducing the run time. Example MS parameters comparing appropriate values for microflow LC and analytical flow LC are detailed in Table 3; the data collected by this method was processed using MultiQuant software 3.0.
The results of this experimental method were compared to those from previously developed analytical flow MS methods for desmopressin and trastuzumab emtansine, respectively. To allow this comparison, the analytical flow methods were performed using the same sample injection volumes as the microflow method, but with a flow rate that was two orders of magnitude faster.
Improving analysis of a small-molecule drug
Desmopressin is used to treat a variety of disorders affecting urine production, including diabetes insipidus and juvenile bedwetting, as well as blood-clotting disorders such as hemophilia A. The drug is generally administered in µg or low-mg doses, and clinical monitoring is performed by analyzing plasma concentrations. The low doses combined with high plasma matrix effects result in some difficulty for routinely monitoring a drug classified by the World Health Organization as an “essential medicine.”1 Although LC/MS analysis methods for desmopressin have been reported,2 there remains a need for highly sensitive methods that reduce sample volume. Desmopressin was therefore analyzed by Trap-Elute MicroLC-MS, and results were compared to a previously developed subpicogram quantitation method using conventional flow rates.3
The results obtained demonstrate the value of utilizing microflow LC. The LLOQ for desmopressin in both the microflow and analytical flow methods was 0.5 pg/mL in human plasma. However, the microflow method only required 300 µL of human plasma sample to achieve this LLOQ, less than a third of the amount required by the analytical flow method (1 mL). The method also demonstrated good selectivity; there was no interference detected from the blank matrix controls. The calibration curve, shown in Figure 1, covered three orders of magnitude over the entire range of plasma samples tested (0.5–250 pg/mL), and displayed good linearity (r = 0.997). The accuracy of the method across the concentrations tested was found to be 91.82–104.34%, surpassing FDA bioanalysis guidelines.
Figure 1 – Calibration curve for quantitation of desmopressin in human plasma (0.5–250 pg/mL); r = 0.997.
In order to compare sensitivity between the new microflow LC method and the analytical flow LC method, the same set of samples was analyzed using the same injection volumes. As shown in Figure 2, the microflow LC enhanced the signal-to-noise ratio by giving more distinct peaks at low desmopressin concentrations, while requiring significantly less sample. Additional benefits of this method are its accessibility and ease-of-use. The setup used requires no special tools for column connections and no manual adjustments for optimized spray, making it less labor-intensive than other microflow LC methods.
Figure 2 – Extracted ion chromatograms of desmopressin (a, b) and desmopressin-d5 internal standard (c, d) at 2.5 pg/mL. Results show a visual comparison of sensitivity between microflow LC (a, c) and analytical flow LC (b, d).
Applying the workflow to a large-molecule drug
Trastuzumab emtansine is an ADC approved for use in HER2-positive metastatic breast cancer. The drug must be monitored during development across complex biofluids at low concentrations. To achieve this, scientists require a robust, reproducible, and exceptionally sensitive workflow that is easy to implement. Analytical flow LC methods often deliver insufficient sensitivity for this, while previous microflow methods have been too slow and have not been user friendly. The Trap-Elute microflow LC method was applied to trastuzumab emtansine to explore the applications and capabilities of the workflow. As with desmopressin, trastuzumab emtansine was analyzed by both Trap-Elute microflow LC and analytical flow LC for comparing sensitivity and sample consumption. Sample preparation utilized an innovative immunoaffinity technique that produced a clean sample and minimized baseline interference.4
Signal-to-noise using microflow LC was improved fourfold over analytical flow LC/MS analysis, using a total sample volume of only 25 µL plasma (Figure 3). Additionally, the assay achieved a wide linear dynamic range of 1–20,000 ng/mL (Figure 4) with accuracy of between 87 and 109%. Most significantly, LLOQ was improved fivefold over conventional methods with a value of 1 ng/mL, underscoring the value of the method for quantifying the increasing number of biologics. The results demonstrate the significant sensitivity gains that can be achieved by utilizing microflow LC and, by using the Trap-Elute workflow, large sample volumes can be loaded at high speeds. This reduced overall sample-loading times by a factor of 10 over previous microflow LC methods.
Figure 3 – Extracted ion chromatograms (XICs) of a selected multiple reaction monitoring (MRM) transition for trastuzumab emtansine at 2 ng/mL (top) and 5 ng/mL (bottom). Left: XICs generated with analytical LC flow rate; right: XICs generated with microflow LC flow rate. Figure 4 – Calibration curve for quantitation of trastuzumab emtansine in mouse plasma over a concentration range covering 4.5 orders of magnitude; r = 0.996.
As biologics continue to increase as a proportion of commercial drugs, new methods of analysis that use smaller sample volumes and show exceptional sensitivity are critical. The method described has been shown to significantly improve analysis of the small-molecule drug desmopressin as well as the ADC trastuzumab emtansine, and can be extended to many biologics and synthetic peptides due to the system’s versatility in terms of column choice. On the Trap-Elute MicroLC-MS system, the use of fingertight fittings and an innovative source that does not require adjustments results in methods that are accessible to those in even the busiest research labs. This microflow LC workflow allows researchers to reduce sample waste and greatly reduce the production of potentially hazardous solvent waste.
Tyler Davis is product manager at SCIEX, 1201 Radio Rd., Redwood City, CA 94065, U.S.A.; tel.: 210-801-1172; e-mail: [email protected]; www.sciex.com. The technology mentioned herein is for research use only, not for use in diagnostic procedures (RUO-MKT-19-9148-A). Note: AB Sciex is operating as SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license. ©2019 AB SCIEX.