Method and kit for detecting plasma kidney quality by using liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of liquid chromatography-tandem mass spectrometry detection, and provides a method and a kit for detecting plasma kidney quality by using liquid chromatography-tandem mass spectrometry. The invention provides a mass spectrum method for plasma sample kidney quality detection for the first time, a pretreatment method of immunomagnetic bead capture is adopted for sample enrichment, and the detection sensitivity can be improved. Isotopes N15 and C13 are adopted to mark a rennin specific peptide fragment as an internal standard, and method calibration is carried out according to the ratio of the peak area of the rennin specific peptide fragment to the peak area of the internal standard and the concentration. The method is relatively good in precision, and the repeatability and the inaccuracy CV average lt in a laboratory are relatively good; the method and the kit for detecting the plasma kidney quality based on liquid chromatography-tandem mass spectrometry provided by the invention can be popularized and applied in clinical detection.

Description

Technical Field

[0001] This invention relates to the field of liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection technology, and more specifically, to a method and kit for detecting plasma renin mass using LC-MS / MS. Background Technology

[0002] Primary aldosteronism is a common cause of secondary hypertension, characterized by excessive aldosterone secretion from the adrenal cortex, leading to sodium retention, potassium excretion, increased blood volume, and suppression of the renin-angiotensin system. Clinically, it primarily manifests as hypertension accompanied by hypokalemia. Primary aldosteronism subtyping influences treatment selection, requiring clinicians to rely on imaging to determine the lesion type and to make a comprehensive judgment based on biochemical indicators, imaging findings, and bilateral adrenal venous sampling (AVS) results. Renin, as a core regulator of the renin-angiotensin system (RAS), is directly measured, making it the preferred method for most laboratories. Currently, immunoassay is the primary method, with no mass spectrometry method available. While immunoassay methods exhibit less variability and reactivity, they still suffer from greater variability and cross-reactivity compared to mass spectrometry. Therefore, it is necessary to develop a stable and efficient mass spectrometry method for renin mass determination. Summary of the Invention

[0003] The purpose of this invention is to provide a method and kit for detecting plasma renin quality using liquid chromatography-tandem mass spectrometry.

[0004] To achieve the objective of this invention, in a first aspect, this invention provides a method for detecting plasma renin mass using liquid chromatography-tandem mass spectrometry, comprising the following steps: A. Prepare standard solutions of renin at different concentrations; B. Add magnetic beads to the standard solution for extraction and incubation. First, wash the magnetic beads with washing buffer, then elute with elution buffer. Add ammonia, isotope internal standard, and trypsin solution to the collected eluent and incubate for a period of time for enzyme digestion. Collect the enzyme-digested sample for liquid chromatography-tandem mass spectrometry detection. Finally, plot a standard curve with the concentration of the standard solution as the x-axis and the ratio of the peak area of ​​the standard solution to the peak area of ​​the isotope internal standard as the y-axis. C. Replace the standard solution in step B with the plasma sample to be tested, and perform the test using the same method; D. Based on the test results of the plasma sample to be tested, and by referring to the standard curve, obtain the concentration of renin in the plasma sample to be tested; The isotopic internal standards are renin-specific peptides labeled with N15 and C13, including quantitative and qualitative internal standards. The renin-specific peptide corresponding to the quantitative internal standard is LIKTGVWQIQMK(N15,C13)GVS, in which K is lysine labeled (N15,C13); the renin-specific peptide corresponding to the qualitative internal standard is GRVTPIF(C13,N15)DNIISQGVLKED, in which F is phenylalanine labeled (C13,N15).

[0005] Furthermore, in step A, a standard solution is prepared using bovine serum.

[0006] Further, step B specifically involves: adding 40-200 μL of magnetic bead solution to 100-600 μL of standard solution, mixing, and incubating with shaking at 37°C for 30-90 min. Then, washing 1-5 times with 50-200 μL of washing buffer, followed by elution with 20-100 μL of elution buffer. Next, adding 2-6 μL of 5-15% ammonia, 2-20 μL of mixed internal standard solution, and 5-20 μL of 10-50 ng / μL trypsin solution to the collected eluent; incubating with shaking at 37°C for 10-30 h for enzyme digestion, and collecting the sample for instrumental analysis. The mixed internal standard solution consists of a quantitative internal standard of 1-50 ng / mL and a qualitative internal standard of 1-200 ng / mL. The reagent used to prepare the isotope internal standard solution is a 10-100 mM NH4HCO3 solution.

[0007] Preferably, step B is as follows: add 100 μL of magnetic bead solution to 400 μL of standard solution, mix, and incubate at 37°C with shaking for 1 h. Then wash three times with 100 μL of washing buffer, followed by elution with 50 μL of elution buffer. Then add 3.4 μL of 10% ammonia water, 10 μL of mixed internal standard solution (25 ng / mL quantitative internal standard and 125 ng / mL qualitative internal standard), and 15 μL of 16 ng / μL trypsin solution to the collected eluent. Incubate at 37°C with shaking for 24 h for enzyme digestion, and collect the sample for instrumental detection. The reagent used to prepare the isotope internal standard solution is 50 mM NH4HCO3 solution.

[0008] Preferably, the liquid chromatography column used in step B is: ACQUITY UPLC HSS T3 1.8μm, 2.1mm×50mm.

[0009] Preferably, the liquid chromatography detection conditions are: flow rate 0.3 mL / min, column temperature 40 °C.

[0010] Preferably, gradient elution is performed according to the following procedure:

[0011] Preferably, mobile phase A: 0.1% v / v Formic acid aqueous solution, mobile phase B: methanol.

[0012] Preferably, the conditions for mass spectrometry detection in step B are as follows: the mass spectrometer is a Waters Xevo TQ-S, using an electrospray ionization source, positive mode acquisition, capillary voltage 2.5 kV, ion source temperature 150℃, desolventizing temperature 550℃, desolventizing gas: 1100 L / Hr, and cone gas: 150 L / Hr.

[0013] Preferably, the characteristic peptide generated in the mass spectrometer for quantitative detection of renin is LIKTGVWQIQMKGVS (SEQ ID NO:1), with corresponding ion pairs of 545.79→833.43 or 546→833; the characteristic peptide for qualitative detection of renin is GRVTPIFDNIISQGVLKED (SEQ ID NO:2), with corresponding ion pairs of 822.47→722.41.

[0014] Preferably, the ion pairs generated in the mass spectrometer for quantitative detection of the isotope internal standard are: 549.79→841.44 or 550→841.44; and the ion pairs for qualitative detection of the isotope internal standard are: 827.49→727.46.

[0015] Preferably, the cleaning solution used in step B is TBS Tween diluted 20 times with distilled water. TM -20 cleaning solution; the formula for TBS Tween-20 cleaning solution is: 25mM Tris, 0.15M NaCl, 0.05% v / v Tween TM -20; The eluent is Pierce TM IgG Elution Buffer (pH=2) Acidic elution solution.

[0016] Using the method of this invention, the linear detection range of renin in the plasma sample to be tested is 0.01~4 ng / mL.

[0017] The sample to be tested can also be a patient's blood sample, etc.

[0018] Secondly, the present invention provides a kit for detecting renin by liquid chromatography-tandem mass spectrometry, the kit comprising at least one of renin standard solution, isotope internal standard, magnetic beads, washing solution, elution solution, etc.

[0019] The isotopic internal standards are renin-specific peptides labeled with N15 and C13, including quantitative and qualitative internal standards. The renin-specific peptide corresponding to the quantitative internal standard is LIKTGVWQIQMK(N15,C13)GVS, in which K is lysine labeled (N15,C13); the renin-specific peptide corresponding to the qualitative internal standard is GRVTPIF(C13,N15)DNIISQGVLKED, in which F is phenylalanine labeled (C13,N15).

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) This invention provides for the first time a mature method for the detection of renin in plasma samples that can be rapidly applied to clinical practice. It uses an immunomagnetic bead capture pretreatment method to enrich the sample and improve the detection sensitivity.

[0021] (ii) Isotope labeling was used as an internal standard to correct potential matrix effects in mass spectrometry. Screened specific peptides were used as monitoring targets. The peak area ratio of the peptides to the internal standard and the response concentration were used for calibration. The quantitative and qualitative ion pairs were used for the first time.

[0022] (III) This method has good precision, with repeatability and in-laboratory imprecision CV both <15%, and can be widely used in clinical testing. Attached Figure Description

[0023] Figure 1 This is a chromatogram of the synthesized internal standard in a preferred embodiment of the present invention.

[0024] Figure 2 This is a mass spectrum of the synthesized internal standard in a preferred embodiment of the present invention.

[0025] Figure 3 This represents the linear detection range of renin in a preferred embodiment of the present invention.

[0026] Figure 4 This is a typical chromatogram of renin (top) and quantitative internal standard (bottom) in a preferred embodiment of the present invention.

[0027] Figure 5 The effects of different mobile phases and acidity on chromatographic peaks are shown in the preferred embodiment of the present invention.

[0028] Figure 6 The effect of different acidities on chromatographic peaks is shown in a preferred embodiment of the present invention. Detailed Implementation

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] The magnetic beads used in the following examples were purchased from New Industries Biotechnology.

[0031] Example 1: Reagent kit and detection method for detecting renal quality using liquid chromatography-tandem mass spectrometry 1. Preparation of standards: (1) Prepare bovine serum solution: used for the preparation of standards.

[0032] (2) Dissolve 1 mg of renin standard in 1 mL of bovine serum solution to prepare a 1 mg / mL stock solution; then use bovine serum solution to prepare a 0.01 mg / mL standard solution as a stock solution; finally use bovine serum solution to dilute the standard to 20 ng / mL.

[0033] (3) Preparation of standard solutions (Table 1): Table 1 Preparation of standard solutions

[0034] 2. Synthesis and preparation of internal standards (1) Sequence synthesized from internal standards: The renin-specific polypeptide corresponding to the quantitative internal standard is LIKTGVWQIQMK(N15,C13)GVS, in which K is labeled with lysine (N15,C13); the renin-specific polypeptide corresponding to the qualitative internal standard is GRVTPIF(C13,N15)DNIISQGVLKED, in which F is labeled with phenylalanine (C13,N15).

[0035] (2) The chromatogram of the synthesized internal standard is shown in [reference needed]. Figure 1 .

[0036] (3) The mass spectrum of the synthesized internal standard is shown in [reference needed]. Figure 2 .

[0037] (4) Preparation of internal standard solution: Dissolve 1 mg of the quantitative internal standard in 1 mL of 50 mM NH4HCO3 to prepare a 1 mg / mL quantitative internal standard solution. Aliquot the solution and store at -80°C as the stock solution for the quantitative internal standard. Then, prepare a 1 μg / mL quantitative internal standard solution using 50 mM NH4HCO3 as the stock solution for the quantitative internal standard.

[0038] Dissolve 5 mg of qualitative internal standard in 1 mL of 50 mM NH4HCO3 to prepare a 5 mg / mL qualitative internal standard solution, aliquot it, and store it at -80°C as a stock solution for qualitative internal standard. Then, prepare a 5 μg / mL qualitative internal standard solution using 50 mM NH4HCO3 as a stock solution for qualitative internal standard.

[0039] Finally, the 1 μg / mL quantitative internal standard stock solution and the 5 μg / mL qualitative internal standard stock solution were further mixed and diluted with 50 mM NH4HCO3 to form a mixed internal standard solution of 25 ng / mL quantitative internal standard and 125 ng / mL qualitative internal standard for sample pretreatment.

[0040] 3. Liquid Chromatography Detection Method The liquid chromatography column used was: ACQUITY UPLC HSS T3 1.8µm 2.1mm×50mm.

[0041] (1) Mobile phase A is 0.1% v / v Formic acid aqueous solution; mobile phase B is methanol.

[0042] (2) The flow rate and gradient of the mobile phase are shown in Table 2. The flow rate is 0.3 mL / min, the column temperature is 40℃, and the running time is 6 minutes.

[0043] Table 2 Liquid Chromatography Conditions

[0044] 4. Mass spectrometry detection method Mass spectrometry was performed using a Waters Xevo TQ-S system with an electrospray ionization source in positive mode. The capillary voltage was 2.5 kV, the ion source temperature was 150 °C, the desolventizing temperature was 550 °C, the desolventizing gas flow rate was 1100 L / Hr, and the cone gas flow rate was 150 L / Hr. Masslynx software was used for data acquisition and qualitative and quantitative analysis.

[0045] Based on the properties of the compound, select the appropriate acquisition mode, specifically: positive ion, ESI, and MRM modes (Table 3).

[0046] Table 3 Mass spectrometry detection conditions

[0047] Note: Renin internal standard-1: GRVTPIF (F-phenylalanine labeled C13, N15) DNIISQGVLKED; Renin internal standard-2: LIKTGVWQIQMK (K-lysine labeled N15, C13) GVS.

[0048] 5. Optimization of preprocessing methods 5.1 Optimization of Magnetic Bead Volume: The capture effect of different magnetic bead volumes was compared. Different volumes of magnetic beads were added to the same system, and the capture efficiency of different amounts of magnetic beads in standards and plasma was compared (Tables 4 and 5). Using the external standard method, the results from both methods were combined, and it was found that 100 μL of magnetic beads achieved the best capture effect. Therefore, 100 μL of magnetic beads was ultimately selected for the experiment.

[0049] Table 4. Capture efficiency (peak area) of different magnetic bead contents in the standard.

[0050] Table 5. Capture efficiency (peak area) of different magnetic bead contents in plasma.

[0051] 5.2 Optimization of Enzyme Digestion Conditions 1) pH optimization of the enzyme digestion system: The enzyme digestion efficiency was investigated under different pH values. The enzyme digestion system with the addition of 3.0~3.4 μL of 10% ammonia (pH=8.2) had the highest enzyme digestion efficiency (Table 6).

[0052] Table 6 Enzyme digestion efficiency at different pH values

[0053] 2) Optimization of enzyme volume: The enzyme digestion efficiency of enzymes of the same mass but different volumes was compared. Considering all factors, 15 μL of enzyme was selected for the experiment (Table 7).

[0054] Table 7 Enzyme digestion efficiency (peak area) for the same amount of enzyme in different volumes

[0055] 3) Optimization of enzyme concentration: To compare the effect of different enzyme amounts on the digestion effect, the same volume of enzyme but different concentrations were added to parallel tubes of the same treatment. 16 ng / μL of enzyme showed the best digestion effect, so an enzyme concentration of 16 ng / μL was selected for the experiment (Table 8).

[0056] Table 8 Enzyme digestion efficiency (peak area) for different enzyme amounts

[0057] 4) Optimization of enzyme digestion time: Considering the impact of different digestion times on the digestion effect, the differences in digestion effect at different digestion times were investigated. It was found that the best effect was achieved with digestion time of 20-24 hours. The peak areas of the analytes at different digestion times are shown in Table 9.

[0058] Table 9. Optimization of enzyme digestion time (peak area)

[0059] The final sample preprocessing method determined is as follows: Add 100 μL of magnetic bead solution to 400 μL of sample / standard solution and mix. Incubate at 37°C with shaking for 1 h. Then wash three times with 100 μL of washing buffer, followed by elution with 50 μL of elution buffer. Add 3.4 μL of 10% ammonia, 10 μL of mixed isotope internal standard solution (25 ng / mL quantitative internal standard and 125 ng / mL qualitative internal standard) and 15 μL of 16 ng / μL trypsin solution to the elution buffer. Incubate at 37°C with shaking for 24 h for enzyme digestion. Collect the sample for instrumental analysis.

[0060] 6. Performance Verification 6.1 Linearity Validation Renin showed good linear correlation within the linear range of 0.01–4 ng / mL. r >0.990 ( Figure 3 ).

[0061] 6.2 Precision Three concentration samples (low, medium, and high) were prepared, and each sample was tested five times. Five batches were tested consecutively, and the repeatability and in-laboratory imprecision were calculated to be 2.75% to 12.4%.

[0062] 6.3 Recovery rate The recovery rate was verified by using a spiked recovery method, and the recovery rate was 82.71%~104.33% (Table 10).

[0063] Table 10 Spiked Recovery Methods and Experimental Results

[0064] 6.4 Typical Chromatograms Typical chromatograms of renin and internal standard are as follows: Figure 4 As shown.

[0065] 7. Sensitivity test To test the sensitivity of this method, samples with concentrations of 0.01, 0.02, and 0.04 ng / mL were selected and tested 10 times consecutively for each concentration. The CV was calculated and found to be within 20% for all samples (Table 11).

[0066] Table 11 Sensitivity Assessment

[0067] Example 2: Optimization of Detection Conditions Regarding the selection of chromatographic columns, this invention focuses on investigating different stationary phases (including ACQUITY UPLC® Peptide BEH C18 column (300 Å, 1.7 μm, 2.1 mm × 100 mm, Waters Corporation, Milford, MA, USA) and ACQUITY HSS T3 column (1.8 μm, 2.1 mm × 50 mm, Waters Corporation, Milford, MA, USA)). Neither column significantly affected peak shape or quantification results; therefore, the ACQUITY UPLC® Peptide BEH C18 column can be used as an alternative to the ACQUITY HSS T3 column, examining its impact on the retention behavior and peak shape of the target peptide. Methanol and acetonitrile were tested as the B phase in the mobile phase, and the signal response intensity was compared when different concentrations of formic acid solution were added to the mobile phase. Simultaneously, different gradient elution programs were compared to optimize peak shape, response intensity, and the resolution of adjacent interfering peaks to the target peak.

[0068] In the initial stage, the mobile phase was optimized. Methanol and acetonitrile were tried as organic phases, and the proportion of formic acid they contained was optimized. The results of using acetonitrile (phase B) - 0.1% formic acid water (phase A), methanol (phase B) - 0.1% formic acid water (phase A), and methanol (phase B) - 0.15% formic acid water (phase A) were compared. Figure 5 The results showed that the peptides to be tested were poorly retained when acetonitrile was used as the mobile phase, and all were eluted within about 0.5 min. Therefore, acetonitrile was not used. A comparison of 0.1% formic acid solution and 0.15% formic acid solution showed little difference, with a slightly higher peak response when 0.1% formic acid solution was used as phase A. Therefore, methanol was the optimal choice as the organic phase (phase B), and -0.1% formic acid solution as phase A.

[0069] Subsequently, the intensity and shape of the standard chromatographic peaks were compared using 0.1% formic acid in methanol (phase B) - 0.1% formic acid in water (phase A), 0.1% formic acid in methanol (phase B) - water (phase A), and methanol (phase B) - 0.1% formic acid in water (phase A), respectively. The results are shown in [Figure 1]. Figure 6 Under the same gradient conditions, the peak shape and peak intensity are best when the mobile phase is methanol (phase B) - 0.1% formic acid water (phase A). Adding formic acid to the organic phase cannot enhance the peak response.

[0070] Then, under the same mass spectrometry conditions and mobile phase, the same sample was detected using two columns: an ACQUITY UPLC® Peptide BEH C18 column (300 Å, 1.7 μm, 2.1 mm × 100 mm, Waters Corporation, Milford, MA, USA) and an ACQUITY HSS T3 column (1.8 μm, 2.1 mm × 50 mm, Waters Corporation, Milford, MA, USA). The effects of the two detection results on the retention behavior and peak shape of the target peptide were observed. It can be seen that although the two columns have different retention times for peptides, they can both ensure good peak shapes. The ACQUITY UPLC® Peptide BEHC18 column can be used as an alternative to the ACQUITY HSS T3 column.

[0071] The final LC-MS / MS conditions were determined as follows: Liquid phase separation was performed using a Waters Acquity I-Class UPLC ultra-high performance liquid chromatography system, with an ACQUITY UPLC HSS T3 1.8 μm column (2.1 mm × 50 mm), and the column temperature was maintained at 40℃. The mobile phase contained 0.1% [unspecified ingredient]. v / v The formic acid aqueous solution (phase A) and methanol (phase B) were used at a flow rate of 0.3 mL / min, with an injection volume of 10 μL. The optimized gradient elution program was as follows: initially maintained at 65% phase A for 0.5 min, then the gradient changed to 40% phase A within 3.00 min, then to 10% phase A within 4.00 min and held for 4.50 min, finally returning to the initial conditions within 5.00 min. The total analysis time for a single sample was 6.0 min. Under this gradient condition, the target peptide showed no interfering peaks and good peak symmetry.

[0072] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting plasma renin mass using liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: A. Prepare standard solutions of renin at different concentrations; B. Add magnetic beads to the standard solution for extraction and incubation. First, wash the magnetic beads with washing buffer, then elute with elution buffer. Next, add ammonia, isotope internal standard, and trypsin solution to the collected eluent and incubate for enzyme digestion. Collect the enzyme-digested sample for liquid chromatography-tandem mass spectrometry detection. Finally, plot a standard curve with the concentration of the standard solution as the x-axis and the ratio of the peak area of ​​the standard solution to the peak area of ​​the isotope internal standard as the y-axis. C. Replace the standard solution in step B with the plasma sample to be tested, and perform the test using the same method; D. Based on the test results of the plasma sample to be tested, and by referring to the standard curve, obtain the concentration of renal quality in the plasma sample to be tested; The isotopic internal standards are renin-specific peptides labeled with N15 and C13, including quantitative and qualitative internal standards. The renin-specific peptide corresponding to the quantitative internal standard is LIKTGVWQIQMKGVS, where K-lysine is used to label N15 and C13. The renin-specific peptide corresponding to the qualitative internal standard is GRVTPIFDNIISQGVLKED, where F-phenylalanine is used to label C13 and N15.

2. The method according to claim 1, characterized in that, In step A, a standard solution is prepared using bovine serum.

3. The method according to claim 1, characterized in that, Step B is as follows: Add 40-200 μL of magnetic bead solution to 100-600 μL of standard solution and mix. Incubate at 37°C with shaking for 30-90 min. Then wash 1-5 times with 50-200 μL of washing buffer, followed by elution with 20-100 μL of elution buffer. Add 2-6 μL of 5-15% ammonia, 2-20 μL of mixed internal standard solution, and 5-20 μL of 10-50 ng / μL trypsin solution to the collected eluent. Incubate at 37°C with shaking for 10-30 h for enzyme digestion. Collect the sample for instrumental analysis. The mixed internal standard solution consists of a quantitative internal standard of 1-50 ng / mL and a qualitative internal standard of 1-200 ng / mL. The reagent used to prepare the isotope internal standard solution is a 10-100 mM NH4HCO3 solution.

4. The method according to claim 1, characterized in that, The liquid chromatography column used in step B is: ACQUITY UPLCHSS T3 1.8μm, 2.1mm×50mm; The liquid chromatography detection conditions were: flow rate 0.3 mL / min, column temperature 40℃; Perform gradient elution using the following procedure: ； Mobile phase A: 0.1% v / v Formic acid aqueous solution, mobile phase B: methanol.

5. The method according to claim 1, characterized in that, The conditions for mass spectrometry detection in step B are as follows: the mass spectrometer is a Waters Xevo TQ-S, using an electrospray ionization source, positive mode acquisition, capillary voltage 2.5 kV, ion source temperature 150℃, desolvation temperature 550℃, desolvation gas: 1100 L / Hr, and cone gas: 150 L / Hr.

6. The method according to claim 1, characterized in that, The characteristic peptide generated by mass spectrometry for quantitative detection of renin is LIKTGVWQIQMKGVS, with corresponding ion pairs of 545.79→833.43 or 546→833; the characteristic peptide for qualitative detection of renin is GRVTPIFDNIISQGVLKED, with corresponding ion pairs of 822.47→722.

41.

7. The method according to claim 1, characterized in that, The ion pairs generated in the mass spectrometer for quantitative detection of the isotope internal standard are: 549.79→841.44 or 550→841.44; the ion pairs for qualitative detection of the isotope internal standard are: 827.49→727.

46.

8. The method according to any one of claims 1-7, characterized in that, The linear detection range for renin in plasma samples is 0.01–4 ng / mL.

9. A kit for detecting renin by liquid chromatography-tandem mass spectrometry, characterized in that, The kit includes at least one of the following: renin standard solution, isotope internal standard, magnetic beads, washing solution, and elution solution; The isotopic internal standards are renin-specific peptides labeled with N15 and C13, including quantitative and qualitative internal standards. The renin-specific peptide corresponding to the quantitative internal standard is LIKTGVWQIQMKGVS, where K-lysine is used to label N15 and C13. The renin-specific peptide corresponding to the qualitative internal standard is GRVTPIFDNIISQGVLKED, where F-phenylalanine is used to label C13 and N15.

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