(W1530-02-11) Evaluation of API 7500 Integrated System with Microflow LC System M5 Using Sulfamethazine (SDX) Neat Solution

Purpose: It is reported that Microflow LC system can help improve the method sensitivity by coupled to triple quadruple Mass Spectrometer. In this poster, the most sensitive triple quadruple mass spectrometer from Sciex API 7500 is coupled with Microflow LC system M5 and the sensitivity improvement using Sulfamethazine neat solution will be evaluated compared to standard LC system. The recommendation will also be presented regarding to the use of microflow LC system for the quantitation in the regulated bioanalysis.

Methods: Standard Analytical Flow Liquid Chromatography: A Shimadzu Nexera Prominence HPLC system was used for the analytical flow portion of the analysis. The column used was a Gemini-NX 5 µm C18 100 Å, 50 x 2 mm column (Phenomenex). Mobile phase A =100% 0.1% formic acid in water, Mobile phase B=100% 0.1% formic acid in ACN. The gradient program is shown in Table 1-1. The total flow is 0.5 mL/minute and injection volume is 1 µL and 10 µL. Microflow Liquid Chromatography M5: A SCIEX M5 MicroLC-TE was used in direct injection mode, in combination with a source mounted column oven (SCIEX). A Kinetex 2.6 µm XB-C18 100 Å, 150 x 0.5 mm analytical column (Phenomenex) was used. Mobile phase A =100% 0.1% formic acid in water, Mobile phase B=100% 0.1% formic acid in ACN. The gradient program is shown in Table 1-1. The total flow is 20 µL/minute and injection volume is 1 µL. The sample loop is 50 µL. The standard injection mode was used, in which the sample loop remains in the flow path during acquisition. Therefore, 2 more minutes were added before and after the analytical method gradient to fill in the injection loop volume with the initial mobile phase. Mass Spectrometry: A SCIEX API 7500 with IonDrive Turbo V source was used. For the microflow LC experiments, the standard probe and E-Lens were replaced with micro probe and micro E-Lens. MS source parameters were optimized for analytical and microflow and CE, EP, and CXP were kept constant for both analytical flow and microflow LC experiments. The source, gas and MS parameters are listed in Table 1-2.
Sciex OS 3.1.6 software was used for data analysis with MQ4 peak integration algorithm. Area against net solution concentration is regressed.

Results: Standard Analytical HPLC results with injections 1 µL and 10 µL are shown in Table 2-1 and Table 2-2, respectively. Microflow LC results with 1 µL injection are shown in Table 2-3. The LLOQ chromatograms are shown in Figure 1. For the same LLOQ sample, The peak height for Microflow LC is about 10 times of analytical HPLC method with 1 µL injection (Table 3). With the same injection volume, about 10 fold sensitivity improvement can be obtained using Sulfamethazine neat solution with microflow LC system. LLOQ peak height with 1 µL injection using microflow LC is equivalent to analytical HPLC with 10 µL injections (Table 3). The column dilution with large diameter column is observed.

Conclusion: Microflow LC can increase the sensitivity under the same injection volume (1 µL) compared with the traditional HPLC. Sometimes the standard LC with increased sample volume can achieve the similar sensitivity. When sample volume is limited and the sensitivity is needed, it is suggested to use the Microflow LC coupled with mass spectrometer API 7500. However, if separation is needed, it is suggested to use conditional HPLC.

Table 1 Instrument Parameters

Table 2 Experimental Results

Table 3 LLOQ responses and Chromatograms of HPLC and Microflow LC