Rapid method to confirm suitability of laboratory LC systems

Abstract

A simple method for the routine assessment of system performance for liquid chromatographs is presented. The method is a novel use of a commercially available test mix and simple gradient profile to challenge system precision and linearity in order to assess the chromatographic suitability of the system for GxP applications.

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A. Introduction

One of the many challenges analysts face in regulated industries is maintaining the qualification (“suitability”) of their instrumentation. In the pharmaceutical industry, the “readiness” of an instrument is documented in its instrument qualification, operational qualification and performance qualification, known as IQ/OQ/PQ.¹ These qualifications are detailed tests verifying that the instrumentation being used is meeting manufacturer specifications. It is important to note that IQ/OQ/PQ criteria should never exceed manufacturer specifications.² If a company desires tighter specifications, either an alternate vendor should be chosen or the company should work with the vendor(s) to ensure such specifications are within the original design qualification for that instrument.

For chromatographic instrumentation, the IQ/OQ and PQ are initially established at the time of installation. Depending on the usage, the PQ (or equivalent) is re-performed on a scheduled basis. In the interim, pharmaceutical laboratories use method or system suitability criteria to establish the validity of analytical runs.³ Typically, this includes (1) a series of standard injections to establish suitable precision, (2) resolution, tailing and/or plate number determinations to establish suitable chromatographic performance and (3) injection of a practical detection limit or quantification limit standard to establish appropriate sensitivity.

In our laboratory, we have found using the suitability of the various methods we have running at any one time is not a good enough diagnostic tool for LC performance. Several older instruments were running with periodic failures in meeting suitability criteria. This is a significant issue for it entails not only a regulatory issue in quality audits, but incurs significant costs in terms of repeating assays, performing investigations and completing paperwork.

To address this issue, a quick instrumentation-independent suitability method was developed for use as a routine diagnostic tool to investigate instrument performance. The criteria for such a method was (1) be “simple” so that analysts would actually use it and (2) be efficient so minimal cost in analyst and instrument time would be incurred. The final criterion was that the method must be able to detect performance issues so these issues could be addressed prior to the instruments being released for routine GxP testing. An added bonus is that the sequence only takes a few hours or can run overnight. The method presented here meets proposed criteria for UV and fluorescence detection using a commercially available test mix.

B. Reagents

 1. Water – LC Grade or equivalent.

2. Acetonitrile, LC Grade or equivalent.

3. Test Mix, Reversed Phase #2 Test Mix, Phenomenex (Part # ALO-3045). This is a standard test mixture containing Uracil (void volume marker), Acetophenone, Benzene, Toluene and Naphthalene in Acetonitrile.

C. Equipment parameters and conditions

 1. An HPLC system with gradient elution capability, in-line mobile phase degasser, auto-sampler with 2–25 μL injection volume capability and either an ultraviolet absorbance detector, a fluorescence detector or both in series.

2. A data acquisition system.

3. Phenomenex Gemini C18 HPLC column, 100 mm × 4.6 mm, 3 μm (Phenomenex, Part # 00D-4439-E0). (See Tables 1 and 2)

Table 1 Method

1	UV Wavelength		254 nm
	F Wavelength		Ex: 284 nm
			Em: 324 nm
2	Flow Rate:		1 mL min^−1
3	Injection Volume		2–25 μL
4	Column Temperature:		30 °C
5	Mobile Phase	A:	Water
		B:	Acetonitrile

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Table 2 Suitability Gradient Profile

Time (min.)	%A	% B
0.00	80	20
2.00	80	20
20.00	20	80
22.00	20	80
22.10	80	20
Re-equilibrate 10 min.
Run Time: 22 min

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D. Solution preparations

Preparation of test solution

 Prepare a 1 : 100 dilution of the Phenomenex Reversed

Phase #2 Test Mixture using Acetonitrile.

Preparation of needle rinse (if necessary)

 Prepare an 80 : 20 mixture of Acetonitrile/Water.

E. System preparation

 1. Purge injector.

2. Reprime all lines used.

NOTE: Priming of each line should require a minimum of about 20 mL of mobile phase to be drawn to waste as most degassers retain a volume of roughly 15 mL. Adjust this volume as necessary based on the specification of the instrument in question.

3. Allow the system to pump at the initial gradient conditions for at least 30 min, prior to initiating any test runs, to allow the column to equilibrate.

F. System suitability and analysis

 1. Inject Acetonitrile. This injection is intended to complete the equilibration of the system prior to performing the analysis.

2. Make a second injection of Acetonitrile (Blank) and confirm that there are no peaks, other than void volume disturbances, visible in the resulting chromatogram.

3. Make six 10 μL injections of the Test Solution, calculate the %RSD of the peak areas for the six injections. Typical Retention Times are shown in Table 3 and a typical Chromatogram can be seen in Fig. 1.

Table 3 Representative Retention Times for the Test Solution

Analyte	RT/min	RRT
Uracil	1.1 (unretained)	0.12
Acetophenone	8.9	1.00
Benzene	12.3	1.38
Toluene	14.7	1.65
Naphthalene	16.4	1.84

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Fig. 1 Representative Chromatograms of the Test Solution. (A) Uracil, (B) Acetophenone, (C) Benzene, (D) Toluene, (D) Naphthalene. Retention time.

Requirement: The %RSD should be NMT 1.0.

4. Inject Acetonitrile (blank).

Requirement: There should be no significant carryover of any of the peaks present in the Test Solution.

5. Make a 2, 5 and 8 μL injection of the Test Solution. These are the first 3 injections used to determine injector linearity.

6. Make three 10 μL injections of the Test Solution, for use as a bracketing standard.

7. Make a 12, 15, 20 and 25 μL injection of the Test Solution. These are the final 4 injections used to determine injector linearity.

8. Make three 10 μL injections of the Test Solution, for use as a bracketing standard.

9. Perform a linearity calculation using the data from steps 5 and 7. Plot injection volume vs. peak area and calculate the correlation coefficient.

Requirement: The correlation coefficient should be NLT 0.9950.

G. Results and discussion

The procedure is designed so that the system is first challenged to meet system precision requirements of USP.³

After injection of a standard, it is important that the autosampler does not exhibit carryover greater than the practical qualification limit for the LC. This value is typically NMT 0.05% of the nominal method standard. This is where many suitability checks stop. However, systems in use need to be challenged with more than simple precision and carryover.

The procedure moves to challenge not only the linearity of the detector, but the proportionality of the injection system. While it is not critical that an injection value be exactly 10 μL, it is important that the 8 μL and 12 μL injections are 80% and 120% of the nominal 10 uL value respectively. Current LCs on the market today easily post a correlation coefficient of NLT 0.999 from 2–25 uL. For single standard methods, injector precision is more critical than injector accuracy. However, knowing the range of proportionality for each LC injector is an important diagnostic tool as well as documentation of the qualified range in which the instrument can be used in regulated methods. It works as a diagnostic for the detector and autosampler.

The recovery of each standard from the bracketed standard is a better diagnostic of the acceptable injector range than linearity. Each injected volume should be accurate to 98–102% recovery of the theoretical recovery.

In Table 4, the values for a LC system suspected of under performing are presented. Note the high RSD for the precision injections and the poor accuracy of the injections in the linearity run. It should also be noted that correlation is a poor diagnostic tool. Many labs would consider R = 0.99 good enough to illustrate an injector is performing well. Here, the injector is not suitable for GxP chromatography.

Table 4 Suitability Results from an underperforming LC

	Uracil	Acetophenone	Benzene	Toluene	Naphthalene
Rt	1.166	8.249	11.527	14.035	15.706
Rt RSD	0.303	0.411	0.141	0.126	0.189
Std	5.881	253.955	111.047	140.118	178.658
Area RSD	2.864	6.706	6.475	6.592	6.593

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		Uracil	Acetophenone		Benzene		Toluene		Naphthalene
		Area	Area	Recovery (%)	Area	Recovery (%)	Area	Recovery (%)	Area	Recovery (%)
uL	2		37.915	74.6	16.461	74.1	20.839	74.4	26.584	74.4
	5		118.217	93.1	51.639	93.0	65.173	93.0	82.936	92.8
	8		124.067	61.1	54.274	61.1	68.433	61.0	87.191	61.0
	10		241.912	95.3	105.963	95.4	133.587	95.3	170.329	95.3
	12		299.343	98.2	130.712	98.1	164.610	97.9	209.991	97.9
	15		379.953	99.7	166.005	99.7	209.651	99.8	266.993	99.6
	20		512.010	100.8	222.902	100.4	281.141	100.3	358.673	100.4
	25		619.105	97.5	269.649	97.1	340.619	97.2	433.644	97.1
k′		0.2	7.3		10.6		13		14.7
T		1.2	1.1		1		1		1
N		2932	36439		54630		88684		128520
r			0.99224		0.99223		0.99226		0.99220

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The LC instrument from Table 4 was subsequently moved offline for maintenance by the vendor. The vendor determined the autosampler needed to be replaced. After the vendor was finished, the method was again run on this system. The results are listed in Table 5. A significant improvement in precision, accuracy of injection and linearity of response is found. This is the performance expected of GxP instrumentation and with our suitability method, we have documentation that our system is performing at this level.

Table 5 Suitability Results from an acceptable LC

	Uracil	Acetophenone	Benzene	Toluene	Naphthalene
Rt	1.153	8.307	11.599	14.076	15.76
Rt RSD	0	0.34	0.195	0.226	0.206
Std	7.840	351.144	163.360	199.767	251.101
Area RSD	0.384	0.151	0.077	0.001	0.002

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		Uracil	Acetophenone		Benzene		Toluene		Naphthalene
		Area	Area	Recovery (%)	Area	Recovery (%)	Area	Recovery (%)	Area	Recovery (%)
uL	2		71.382	101.6	32.590	99.7	40.445	101.2	51.124	101.8
	5		178.301	101.6	82.815	101.4	101.447	101.6	127.555	101.6
	8		275.660	98.1	128.230	98.1	156.978	98.2	197.619	98.4
	10		351.519	100.1	163.448	100.1	199.765	100.0	251.104	100.0
	12		413.062	98.0	192.760	98.3	235.777	98.4	295.765	98.2
	15		520.729	98.9	242.474	99.0	296.888	99.1	373.074	99.1
	20		689.016	98.1	321.501	98.4	393.271	98.4	493.770	98.3
	25		855.746	97.5	399.462	97.8	489.939	98.1	614.859	97.9
k′		0.2	7.3		10.6		13		14.7
T		1.2	1.1		1		1		1
N		2932	36439		54630		88684		128520
r			0.99993		0.99994		0.99996		0.99995

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Another novel aspect of this method is its ability to challenge the performance of the pumps and gradient proportioning ability by monitoring the resolution of the analyte peaks. Under current evaluation is whether we can use resolution to monitor the pumps and gradient proportioning value.

Fluorescence detection

In the course of this investigation, the need to qualify a fluorescence detector arose. Knowing the test mix contained a fluorophore (naphthalene), the challenge of suitability method was also used with florescence detection. Listed in Table 6, the method illustrated that the fluorescence detector used in series with the UV detector was working suitably for GxP studies.

Table 6 Suitability Results from an acceptable fluorescence detector

	Naphthalene (UV)	Naphthalene (F)
Rt	16.182	16.247
Rt RSD	0.073	0.077
Std	238.847	23.396
Area RSD	0.562	0.716

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		Naphthalene (UV)		Naphthalene
		Area	Recovery (%)	Area	Recovery (%)
uL	2	48.240	101.0	4.742	101.3
	5	118.802	99.5	11.661	99.7
	8	191.061	100.0	18.711	100.0
	12	287.179	100.2	28.034	99.0
	15	360.211	100.5	34.991	99.7
	20	477.044	99.9	46.236	98.8
	25	595.051	99.7	57.697	98.6
r		0.99998		0.99998

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H. Conclusion

The method proposed here is an effective diagnostic tool for routine suitability and diagnostic testing of liquid chromatographs. The method can be used for maintenance or for spot checking LCs such as prior to use for lengthy projects where small changes in performance are not acceptable. In addition, the method can serve as a problem solving tool for validation and CAPA (corrective action/preventative action) investigations.

Notes and references

1. P. Bedson and M. Sargent, Accredit. Qual. Assur., 1996, 1, 265–74.
2. C. Burgess, D. C. Jones and R. D. McDowall, Analyst, 1998, 123, 1879–86.
3. Chromatography: USP General Chapter <621>, US Pharmacopeia 32, 2009, United States Pharmacopeia: Rockville, Maryland 20852-1790, USA.

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