SOP for the Performance Verification of an LC-MS system

OBJECTIVE

This document describes the procedure for the verification of the performance of an LC-MS system through an in-house method. Performance verification is necessary in order to ensure the proper functioning of the LC-MS system. The verification involves the determination of the quality parameters for an LC-MS/MS analysis of naphthyl acetic. The acceptability of the results is assessed by the defined acceptance criteria.

SCOPE

This procedure was established for the performance verification of an LC system coupled to the LCQ-MS using the LC-MS/MS analysis of naphthylacetics (1-naphthyl acetamide, 1-naphthoxyacetic acid and 2-naphthoxyacetic acid) in the selected reaction monitoring mode (SRM). The quality parameters that are determined are as follows: 

1. Limit of detection, 
2. Limit of quantification, 
3. Linearity and range,
4. Precision.

DEFINITIONS

Precision

expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

Limit of detection (LOD)

the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

Limit of quantitation (LOQ)

the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

Linearity 

the ability (within a given range) to obtain test results that are directly proportional to the concentration (amount) of analyte in the sample.

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Range

the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

RESPONSIBILITIES

The analyst must be familiar with the operation of the LC-MS instrument when carrying out this task.

METHOD SUMMARY

The compounds are separated via the RP-amide column and identified and quantified using electrospray ionization mass spectrometry. The LCQ MS detector is operated in the positive mode for 1-naphthylacetamide determination and in the negative mode for naphthoxyacetic acid determination.

The Xcalibur data system is used to control the data acquisition and to store and manipulate the mass spectral data. These compounds are identified and quantitated in the selected reaction monitoring mode (SRM) using the quantification and confirmation ions presented in Table 1.

Analyte	Quantitation ion m/z	Confirmation ion m/z
1-naphthylacetamide	141	169
1-naphthoxyacetic acid	143	157
2-naphthoxyacetic acid	143	157

Table 1. Mass spectral Quantification and confirmation ions.

When using the Dionex HPLC system, instrument control is via the Chromeleon software, whereas, the Waters Alliance LC is controlled manually.

EQUIPMENT AND SUPPLIES

• Dionex HPLC system or Waters Alliance liquid chromatography
• Finnigan LCQ MS detector with Xcalibur data system software
• Column: Ascentis® Express RP-Amide, 10cm x 2.1 mm, 2.7 μm (Supelco)
• Autosampler clear glass vials with screw cap, 1.8 mL, 12 x 32 mm (Thermo)
• Analytical balance (Mettler Toledo or equivalent)
• Pipettor (Socorex)
• Spatula

REAGENTS AND STANDARDS

Reagents

• Methanol, LC-MS grade (Fluka or equivalent)
• Water, LC-MS grade (Fluka or equivalent)
• Glacial acetic acid, analytical grade (Merck or equivalent)

Standards

• 1-naphthylacetamide PESTANAL®, 99% purity (Sigma-Aldrich)
• 1-naphthoxyacetic acid, 98%purity (Aldrich)
• 2-naphthoxyacetic acid PESTANAL®, 98% purity (Sigma-Aldrich)

Preparation of mobile phase and standard solutions

• 2mM acetic acid solution: Add 115 μL of acetic acid to 1000 mL water and mix.
• 1,000 ppm stock solution: Prepare a 1,000 ppm stock solution of each analyte. Weigh 5 mg of the solid standard and dissolve in 5 mL methanol. Weigh the solution. The concentration of the stock solution is calculated as follows:

• 5 ppm 1-NAD standard solution: Pipet 5 μL of 1,000 ppm stock solution of 1-NAD and mix with 995 μL of methanol
• 5 ppm 1-NOA standard solution: Pipet 5 μL of 1,000 ppm stock solution of 1-NOA and mix with 995 μL of methanol.
• 5 ppm 2-NOA standard solution: Pipet 5 μL of 1,000 ppm stock solution of 2-NOA and mix with 995 μL of methanol.
• 500 ppb 1-NAD standard solution: Pipet 100 μL of 5 ppm solution of 1-NAD and mix with 900 μL of methanol.
• 500 ppb 1-NOA standard solution: Pipet 100 μL of 5 ppm solution of 1-NOA and mix with 900 μL of methanol.
• 500 ppb 2-NOA standard solution: Pipet 100 μL of 5 ppm solution of 2-NOA and mix with 900 μL of methanol.

• 10 ppm intermediate standard solution: Pipet 30 μL each of the analyte stock solution and mix with 3.910 mL of methanol. Weigh the aliquot and the solution.
• 200 ppb intermediate standard solution: Pipet 40 µL of the I0 ppm standard solution and mix with 1.960 mL methanol. Weigh the aliquot and the solution.
• Working standard solutions: Prepare the working standard solutions to be used for linearity and LOD determinations as outlined in Table 2. A standard is prepared by taking an aliquot of the intermediate standard solution into a glass vial and adding methanol. The weights of the aliquot and the resulting solution are recorded and used for the calculation of the concentration.

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From 10 ppm intermediate standard solution			From 200 ppb intermediate standard solution
Concentration, ppb	The volume of 10 ppm solution, µL	The volume of methanol, µL	Concentration, ppb	Volume of 200 ppb solution, µL	The volume of methanol, µL
100	10	990	50	250	750
200	20	980	40	200	800
400	40	960	25	125	875
500	50	950	10	50	950
600	60	940	5	25	975
800	80	920	2.5	12.5	987.5
1,000	100	900

Table 2. Preparation of the working standard solutions.

LIQUID CHROMATOGRAPH/MASS SPECTROMETER PREPARATION

Liquid Chromatograph Conditions

The HPLC conditions are as follows:

Column: Ascentis Express RP-Amide, 10 cm x 2.1 mm, 2.7μm, Supelco

Mobile Phase: 2 mM acetic Acid: Methanol

Flow rate: 0.300 mL/min

Column Temperature: 50°C

Injection volume: 5 μL

Time, min	%Methanol	%Acetic acid
0	30	70
2	30	70
4	45	55
5	45	55
8.4	70	30
11.8	70	30
12.6	30	70
16.6	30	70

Table 3. Gradient elution program

Mass Spectrometer Conditions

The mass spectrometer detection parameters are shown in Table 4. The ESI positive mode is run separately from the ESI negative mode.

Table 4. MS detection parameters.

ANALYTE IDENTIFICATION

Prior to the analysis, the elution order of the analytes must be determined and/or verified. This is carried out by injecting a standard solution (500 ppb) containing only the analyte and determining the retention time. The MS/MS spectrum is also acquired.

PROCEDURE

Tuning the MS

Prior to analysis, the mass spectrometer must be tuned to maximize the sensitivity of the instrument. Tuning involves the direct infusion of 5ppm of 1-NAD and 5 ppm of 1-NOA via the syringe pump at a flow rate of 300μL/min and using a 50/50 methanol: 2mM acetic acid mobile phase. The scan parameters used were as follows: Scan mode: MS; Scan type: Full; MSn power: 1; Number of Microscans: 3; Maximum inject time: 100; Input Method: From/To; Source: Off; Scan Fragmentation: F rom Mass 50 to 300. The automatic tuning is done via the Tune program of the instrument.

Determination of quality parameters

1. Instrument limit of detection (LOD) and limit of quantification (LOQ)

• The limit of detection is determined by injecting standard solutions of decreasing concentrations. For this purpose, the standard solution used ranges from 2.5 ppb to 100 ppb.
• After injection, the chromatogram is inspected. The LOD is the concentration of the analyte giving a signal 3 times more than the noise. The LOD is reported as the amount (ng) of analyte injected in the chromatographic run and is derived as follows:

LOD = concentration (ng/ μL) x injection volume (μL)

• The LOQ is reported as the amount (ng) of analyte injected that gives a signal 10 times more than the noise. It is estimated from the LOD data using the expression

LOQ = 3.3 x LOD

2. Linearity

• The linearity is determined over the range that is appropriate to the lowest and the highest nominal concentration permissible in the instrument. The lowest concentration is defined such that is similar to the determined LOQ. 
• Determinations at 5 or more concentrations must be made. Concentrations ranging from 25 ppb to 1 ppm are typically used. A calibration plot (peak area vs. concentration) is constructed and the equation of the calibration line and the correlation coefficient (r) must be reported and the calibration plot is submitted. 
• The linearity of the plot is evaluated in terms of the correlation coefficient (r). A linear plot must have an r ≥ 0.99.

3. Precision (Repeatability)

• The repeatability is determined by performing 6 repeated injections of a standard (near the midrange of the calibration curve). The mean, the standard deviation and %RSD are calculated and the number of determinations is reported. 
• The %RSD is used to evaluate the precision (repeatability) of the method. The acceptability of the % RSD is assessed based on the reproducibility CV (% RSDR) that can be derived using the Horwitz equation which is expressed as

Where 

C is the mass fraction expressed as a power (exponent) of 10.

The repeatability (% RSDr) acceptability is proposed to be the Horwitz value for %RSDr x 0.67.

Peak Area integration

• The Xcalibur data system is used for the integration of the peak area. Automatic peak area integration can be used. However, if during the review of the chromatogram, the analyst notices improper integration by the data system, the peaks can be reintegrated manually.
• Smoothing actions can be applied in the chromatogram in order to minimize the influence of noise. However, use the minimum points for smoothing.

Annexure

Nil

Revision History

Nil

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