The IQ requirements provide evidence that the hardware and software are properly installed in the desired location.

Acceptance criteria for critical instrument parameters that establish “fitness for purpose” are verified during IQ and OQ. Specifications for particular instruments and applications can vary depending on the analytical procedure used and the desired accuracy of the final result. Instrument vendors often have samples and test parameters available as part of the IQ/OQ package.

Wherever possible in the procedures detailed as follows, certified reference materials (CRMs) are to be used in preference to laboratory-prepared solutions. These CRMs should be obtained from a recognized accredited source and include independently verified traceable value assignments with associated calculated uncertainty. CRMs must be kept clean and free from dust. Recertification should be performed periodically to maintain the validity of the certification.

Ensure that the accuracy of the wavelength axis (x-axis) over the intended operational range is correct within acceptable limits.

For non-diode array instruments, wavelength accuracy and precision are determined over the operational range using at least six replicate measurements. For wavelength accuracy, the difference of the mean measured value to the certified value of the CRM must be within ±1 nm in the UV region (200–400 nm), and in the visible region (400–700 nm) must be within ±2 nm. For wavelength precision, the standard deviation of the mean must not exceed 0.5 nm. For diode array instruments, only one wavelength accuracy measurement is required, and no precision determination needs to be performed. The difference between the certified and measured value of the CRM must not exceed ±1 nm in the UV region (200–400 nm), and in the visible region (400–700 nm) must not exceed ±2 nm.

To establish the transmittance accuracy, precision, and linearity of a given system, it is necessary to verify the absorbance accuracy of a system over its intended operational range by using the following procedures as appropriate for the wavelength and absorbance ranges required.

Although the measurement of absorbance or transmittance is a ratio measurement of intensities and therefore theoretically is independent of monochromatic source intensity, practical measurements are affected by the presence of unwanted radiation called “stray radiant energy” or “stray light”. In addition, the adverse effect of stray light increases with aging of optical components and lamps in a spectrophotometer. The effects are greater at the extremes of detector and lamp operational ranges. Analysts must monitor the level of stray light at appropriate wavelength(s) as part of PQ. Stray light can be detected at a given wavelength with a suitable liquid filter. These solutions are available as CRMs or can be prepared at the concentrations shown in Table 3 by using reagent-grade materials.

When using a 5-mm path length cell (filled with the same filter) as the reference cell, and then measuring the 10-mm cell over the required spectral range, analysts can calculate the stray light value from the observed maximum absorbance using the formula:

This procedure simply requires the 10-mm cell measurement to be referenced against the 5-mm cell (filled with the same filter) and therefore can be achieved by either chronological or spatial referencing in any type of spectrophotometer. Alternatively, analysts can measure the absorbance of the filters specified in Table 3 against the appropriate reference, and record the maximum absorbance value. (An S value of 0.01 is produced by an A value of 0.7A, which equates to a maximum absorbance value of 2A measured by this alternate procedure.) [Note—For some instruments where absorbance values greater than 3A cannot be reported directly, this procedure may require a two-step process whereby the sample beam initially is attenuated by a 1- to 2-A filter, the value of which is measured and recorded. After zeroing the instrument with this filter in place, measure the stray-light filter, and again record the absorbance value. The estimated stray light value is now the sum of these two absorbance readings. ]

If accurate absorbance measurements must be made on benzenoid compounds or other compounds with sharp absorption bands (natural half-bandwidths of less than 15 nm), the spectral bandwidth of the spectrophotometer used should not be greater than 1/8th the natural half-bandwidth of the compound's absorption.

Determine the resolution of the spectrophotometer by using the following procedure. Measure the ratio of the absorbance of a 0.020% (v/v) solution of toluene in hexane (UV grade) at the maximum and minimum at about 269 and 266 nm, respectively, using hexane as the reference. The absorbance ratio obtained depends on the spectral bandwidth of the instrument. For most pharmacopeial quantitative purposes, a spectral bandwidth of 2 nm is sufficient, and the acceptance criteria for the ratio is NLT 1.3.

The effect of spectral bandwidth and measurement temperature on the ratio is shown in Table 4.4

Suitable CRMs may also be used for this measurement. Alternatively, a suitable atomic line can be scanned in single-beam mode, and the peak width at half peak height can be determined. This peak width at half peak height equates to the bandwidth of the spectrophotometer.

The purpose of PQ is to determine that the instrument is capable of meeting the user's requirements for all the parameters that may affect the quality of the measurement and to ensure that it will function properly over extended periods of time.

For determinations using UV or visible spectrophotometry, the specimen generally is dissolved in a solvent. Unless otherwise directed in the monograph, analysts make determinations at room temperature using a path length of 1 cm. Many solvents are suitable for these ranges, including water, alcohols, lower hydrocarbons, ethers, and dilute solutions of strong acids and alkalis. Precautions should be taken to use solvents that are free from contaminants that absorb in the spectral region under examination. For the solvent, analysts typically should use water-free methanol or alcohol or alcohol denatured by the addition of methanol but without benzene or other interfering impurities. Solvents of special spectrophotometric quality, guaranteed to be free from contaminants, are available commercially from several sources. Some other analytical reagent-grade organic solvents may contain traces of impurities that absorb strongly in the UV region. New lots of these solvents should be checked for their transparency, and analysts should take care to use the same lot of solvent for preparation of the test solution, the standard solution, and the blank. The best practice is to use solvents that have NLT 40% transmittance (39.9%T = 0.399A) at the wavelength of interest.

Assays in the visible region usually call for concomitantly comparing the absorbance produced by the assay preparation with that produced by a standard preparation containing approximately an equal quantity of a USP Reference Standard. In some situations, analysts can omit the use of a reference standard (e.g., when spectrophotometric assays are made with routine frequency) when a suitable standard curve is available and is prepared with the appropriate USP Reference Standard, and when the substance assayed conforms to the Beer–Lambert law within the range of about 75%–125% of the final concentration used in the assay. Under these circumstances, the absorbance found in the assay may be interpolated on the standard curve, and the assay result can be calculated. Such standard curves should be confirmed frequently and always when a new spectrophotometer or new lots of reagents are put into use.

Validation is required when a UV-Vis method is intended for use as an alternative to the official procedure for testing an official article.

The objective of UV-Vis method validation is to demonstrate that the measurement is suitable for its intended purpose, including quantitative determination of the main component in a drug substance or a drug product (Category I assays), quantitative determination of impurities or limit tests (Category II), and identification tests (Category IV). Depending on the category of the test (see Table 2 in Validation of Compendial Procedures 1225), the analytical method validation process for UV-Vis requires testing for linearity, range, accuracy, specificity, precision, detection limit, quantitation limit, and robustness. These analytical performance characteristics apply to externally standardized procedures and those that use standard additions.

Chapter 1225 provides definitions and general guidance on analytical procedures validation without indicating specific validation criteria for each characteristic. The intention of the following sections is to provide the user with specific validation criteria that represent the minimum expectations for this technology. For each particular application, tighter criteria may be needed in order to demonstrate suitability for the intended use.

Current US Good Manufacturing Practices regulations [21 CFR 211.194(a)(2)] indicate that users of analytical procedures described in USP–NF are not required to validate these procedures if provided in a monograph. Instead, they simply must verify their suitability under actual conditions of use.

The objective of a UV-Vis procedure verification is to demonstrate the suitability of a test procedure under actual conditions of use. Performance characteristics that verify the suitability of a UV-Vis procedure are similar to those required for any analytical procedure. A discussion of the applicable general principles is found in Verification of Compendial Procedures 1226. Verification is usually performed using a reference material and a well-defined matrix. Verification of compendial UV-Vis procedures includes at minimum the execution of the validation parameters for specificity, accuracy, precision, and quantitation limit, when appropriate, as indicated under Validation.USP38