Q Exactive Focus Application of Different High Resolution Quantitative Methods in Pharmaceutical Analysis

1 Introduction

Mass spectrometry has been widely used in drug analysis for its excellent sensitivity and specificity. Traditional mass spectrometry uses the unit resolution to select the ion detection SIM or the selective reaction to monitor the SRM quantitative method. The high resolution mass spectrometry has the exact mass and separation of the compound ions. The ability to adjacent mass peaks is increasingly being used in quantitative analysis.

Based on Orbitrap's electrostatic field orbital technology, Q Exactive Focus combines high-performance quadrupoles with high-resolution Orbitrap technology with resolutions of up to 70,000 FWHM and long-term stable high-quality precision for high-quality primary and secondary High-resolution mass spectrometry data guarantees the reliability of qualitative and quantitative results; comparable to the sensitivity and wide linear range of high-end triple quadrupoles, it is easy to quantify trace components in drugs. Q Exactive Focus offers different high-resolution quantitative modes to meet different quantitative needs:

a) Full Scan (High Scan) High Resolution Quantitation: Compound ions in all scan ranges are passed through the quadrupole and sent to Orbitrap for detection. The XIC spectra are extracted from the Fullscan data by accurate masses for quantification because QExactive Focus has good The quality accuracy, so the extraction window can be reduced to 5 ppm or even lower, which fully guarantees the extremely high selectivity of the method. This quantitative method does not need to predict the compound information, and can record the mass spectrometry signals of all known and unknown compounds. Retrospective analysis of data.

b) Select ion detection (SIM) high-resolution quantification: Selectively pass the target compound through a quadrupole, then send the target compound to Orbitrap for high-resolution scanning, and then quantify the XIC spectrum by first-order accurate mass extraction. This quantitative method can remove a large amount of interfering ions by screening and filtering high-performance quadrupole, so that the target compound can enter the detector more, which improves the sensitivity of detection.

c) Parallel reaction detection (PRM) high-resolution quantification: the selective passage of the target compound by the quadrupole, the high-energy collision fragmentation of the ions entering the collision cell after passing, the generated fragments will be simultaneously sent to Orbitrap for high resolution. Scan and then select high resolution secondary ion to quantify. This quantitative method increases the sensitivity by filtering out a large number of interfering ions through the quadrupole, and the second-order high-resolution mass spectrometer further enhances the quantitative specificity.

In this paper, we quantify the N-(dimethyl adamantane) glycine hydrochloride, and compare the above-mentioned high-resolution quantitative methods. The characteristics of different quantitative methods are explained from the principle, the sensitivity of quantitative effect, Linear ranges and specificities were compared.

Figure 1. Full scan (FullScan) high resolution quantitative schematic

Figure 2. Selective ion detection ( SIM ) high resolution quantitative schematic

Figure 3. Parallel reaction detection ( PRM ) high resolution quantitative schematic

2. Experimental conditions

2.1 Liquid chromatographic conditions:

Instruments: Dionex UltiMate 3000 Ultra Performance LC Column: Thermo Scientific Hypersil Gold aQ (100 x 2.1 mm, 1.9 μm)
Mobile phase: A is the aqueous phase: 0.1% formic acid water, B is the organic phase: acetonitrile gradient conditions:

2.2 Mass spectrometry conditions

Instrument: Thermo Q Exactive Focus Quadrupole - Electrostatic Field Orbitrap High Resolution Tandem Mass Spectrometry Ion Source Parameters: HESI Spray voltage: +3.5 KV; Sheath Gas Pressure: 40 arb; Aux Gas Pressure: 10 arb; Capillary Temp: 300 ° C; Heater Temp: 350°C

2.3 Sample preparation

Take the N-(dimethyl adamantane) glycine reference stock solution of memantine hydrochloride, and dilute it to make N-(dimethyl adamantane) glycine 0.12, 0.6, 1.2, 6.0, 12.0, 60, 120, 600, 1200, 6000, 12000 ng/ml (ppb) solutions were injected and analyzed according to the different scanning modes described above.

3. Results

3.1 Full Scan (FullScan)

Full Scan FullScan quantifies all ion signals entering the detector and then quantifies the target compound mass spectral response by precise molecular weight extraction. Figure 4 shows the extracted ion current signal of the target compound at full concentration of 0.6, 1.2, 6.0 ng/ml. Even at 0.6 ng/ml, the sample loading is only 0.6 pg. The target compound can still be detected by full scan. The signal is clearly visible, the signal intensity is further increased at 1.2 ng/ml, and the extracted ion flow window uses a uniform 5 ppm (0.0012 amu), indicating that QE Focus maintains excellent mass accuracy at very low concentrations.

Investigate the linear range and reproducibility of full scan quantitation. One-step full-scan accurate mass number XC chromatogram of N-(dimethyl adamantane) glycine, 1.2 ng/ml~12 μg/ml standard curve is shown in Figure 5, 1.2 ng/ml and 120 ng/ml The precision of each 5-pin is shown in Figure 6.

Figure 6. Full scan quantitative precision ( 1.2 , 120 ng/ml )

The results showed that N- (dimethyl adamantane) glycine had a good linear relationship at 1.2 ng/ml~12 μg/ml , Y=-138720+362674X-1.44506X 2 , R 2 =1.0000 , and the deviations of each concentration point were in Within 5% . In the limit of quantitation 1.2 ng ml / and high concentrations of 120 ng / ml were good reproducibility, the needle 5 were less than 4% RSD

3.2 Select ion monitoring ( SIM )

Selective Ion Monitoring SIM High Resolution Quantitation The target compound is selectively passed through a quadrupole and the target compound is sent to Orbitrap for high resolution scanning. By screening and filtering the quadrupole, a large amount of interfering ions can be removed, allowing the target compound to enter the detector more and then quantified by one-stage accurate mass extraction. Extraction relatively 0.12 ng / ml SIM mode ion current signal, the AGC value binding for each concentration and the time of ion implantation, it is understood the number of target ions into the compound at Orbitrap detector ml concentration of 0.12 ng / increased significantly. Even if the concentration is reduced by 10 times than the full scan , a better peak shape can be obtained.

However, in the process of quantification, there is often interference of isomers or even isomers, and an effective distinction from the target compound cannot be obtained by the first-order accurate mass. For example, in the SIM mode, each concentration of XIC can be observed for 6.2 min. There is an interference peak ( part of the blue dotted line in Figure 7 ).

Figure 7. Selected ion monitoring chromatograms of extracted ion chromatograms at 0.6 PPb , 1.2 PPb , 6.0 PPb concentrations

It can be seen from the first-order mass spectrometry data in Fig. 8 that there is an interfering ion of m/z 238.014 in the peak of 6.2 min , which is very close to the target impurity N- (dimethyl adamantane) glycine, only 0.5 ppm difference , and cannot pass one. The mass spectrum distinguishes the two.

Figure 8. Mass spectrometry comparison of N- (dimethyl adamantane) glycine and interference peaks

3.3 Parallel Reaction Monitoring ( PRM )

Parallel reaction detection ( PRM ) high-resolution quantification of the target compound through the quadrupole,

The ions enter the collision cell immediately after passing and a high-energy collision fragmentation occurs. The generated fragments are simultaneously sent to Orbitrap for high-resolution scanning, and then the high-resolution secondary product ions are selected for quantification. The quantitative method is characterized in that while the quadrupole filters out a large amount of interfering ions to increase the sensitivity, the two-stage high-resolution mass spectrometer further improves the quantitative specificity.

In order to better perform PRM quantification, it is necessary to select high-quality quantitative daughter ions, and use Q Exactive Focus to perform secondary fragmentation of the target impurity N- (dimethyl adamantane) glycine, and obtain high-quality secondary mass spectrometry. After the figure, it is introduced into the professional small molecule structure analysis software Mass Frontier 7.0 , which can quickly and accurately complete the automatic speculation of different fragment ions and their corresponding structures (Fig. 9 ).

Figure 9. Mass Frontier automatically assigns secondary MS fragments

The software contains the Fragmentation Library fragmentation database, which covers almost all published literature. It can obtain the complete fragmentation mechanism while quickly completing the fragmentation structure, and can conveniently call and view the cited literature to ensure The accuracy of the fragmentation analysis (Figure 10 ).

Figure 10. Fragmentation Library provides fragmentation mechanism

From the above analysis, the fragments of m/z 107.0855 and 163.1481 have higher quality and are the preferred ions for PRM quantification.

Further, by comparing the N- (dimethyl adamantane) glycine secondary mass spectrum and the 6.2 min interference ion secondary mass spectrum (Fig. 11 ), it was found that m/z 107.0855 fragment ions existed in both (blue) The dotted line box), the ion quantification can not effectively distinguish the target compound and the interfering ion, and the m/z 163.1481 fragment ion is only present in the secondary spectrum of the target compound (the red dotted frame part), and the ion quantification is higher. Specificity.

Figure 11. Secondary mass spectrometric alignment of N- (dimethyl adamantane) glycine and interference peaks

By comparing SIM quantitative and quantitative effects PRM, PRM be quantified by using the product ion m / z 163.1481, better removal of background interference, higher specificity (FIG. 12).

Figure 12. Extract ion flow ratios for different quantitative methods and different product ion selections

4. Conclusion:

4.1 This method uses Q Exacitve Focus for high-resolution quantification and obtains excellent quantitative results. The full-scan quantitative linear range was 1.2 ng/ml~12 μg/ml , R 2 =1.000 , and the deviation between each concentration point and linearity was less than 5% , and RSD was less than 4% in both high and low concentrations . Selecting the ion monitoring SIM to filter the background interference by quadrupole and screening the target parent ions for accumulation, the quantitative sensitivity can be increased to 0.12 ng/ml , which is 200 times higher than the import registration standard , and the whole linear range is up to 5 orders of magnitude; parallel reaction monitoring PRM Filtering background interference by quadrupole and screening for the accumulation of target parent ions for high-energy cleavage, thereby obtaining high-quality full-scan information of secondary ion, which can effectively eliminate homogeneity and even isomerism while ensuring sensitivity. Interference to get higher specificity.

4.2 Q Exactive Focus combines a high-performance quadrupole with high-resolution Orbitrap technology with a resolution of up to 70,000 FWHM and long-term stable high-quality accuracy, achieving high quality even at very low levels of impurities. Grade and secondary high-resolution mass spectrometry data ensure the reliability of qualitative and quantitative results; comparable to the sensitivity and wide linear range of high-end triple quadrupoles, it is easy to quantify trace impurities in drugs. Compared with triple quadrupole mass spectrometry, Q Exactive Focus uses first-order mass spectrometry to quantify the precursor ion and sub-ion parameters. It can be accurately quantified by using only the precise molecular mass of the target compound, which is more convenient and faster. By using two-stage high-resolution mass spectrometry, the full scan information of the daughter ions can be recorded, and the more specialized daughter ions can be flexibly selected for quantification in the data post-processing, and the matrix interference can be effectively eliminated, the selectivity is better, and the sensitivity is higher.

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