Utilization of P-Test Verification for Equate Cold Tablets in Comparison in Alka-Seltzer

Raman spectroscopy has been shown to be a useful tool used to distinguish any differences that exist between competing over- the-counter (OTC) cold medicines. Additionally, this analytical tool also provides information on the best practices that should be used in order to establish a training set that will permit detection of miniscule differences between samples. The Metrohm Instant Raman Analyzer (Mira P) is designed for the rapid, nondestructive identification and verification of samples.

The identification of an unknown sample involves comparing of its spectrum within a library of known compounds, which results in the establishment spectral matches based on their similarity. The verification is typically used when the authenticity of a known sample must be confirmed, as this technique especially sensitive to miniscule spectral differences within a sample.

A p-test is a type of multivariate analysis technique that is used for verification purposes, as this technique measures the acceptable variability of a sample spectrum in comparison to a representative training set. The reported value indicates the statistical confidence that the sample aligns most closely to in the training set. Therefore, the spectral data collected by this tool can be used to assess and compare the chemical similarities between both generic and more recognized pharmaceutical drugs.

The Mira P is equipped with Orbital Raster Scan (ORSTM) technology that improves the interrogation area on the sample while simultaneously ensuring a high spectral resolution to allow for even inhomogeneous samples, such as effervescent cold tablets, to be verified with the utmost confidence.

Experimental Data

The effectiveness of a p-value depends on the quality (or robustness) of the training set. A robust training set accounts for normal variations, unrelated to the chemical composition of the sample, which may be encountered in the course of verification. For example, multiple batches from the same manufacturer or batches from different producers should be sampled for the training set.

Additional variances such as ambient light and temperatures, differences in sample containers and any potential instrument variability should also be considered. Spectra in this experiment were collected from different locations on a minimum of twenty different sample tablet to generate a training set that accurately represents the full inhomogeneity of the tablets.

To collect 60 different spectra, an excitation wavelength of 785 nm was used by the Mira P that was equipped with a short working distance (SWD) attachment was used. The 60 different spectra included 20 with either high, low or auto integration settings. These spectra were then processed using MiraCal software. The same operating procedure of the instrument acquisition parameters was also used to establish the training set for the sampling of the tablets.

Laser Power 5
Auto integration ON
Average 1
Smart tip Allow All
Confidence interval 0.95
Match score 0.85
Library USP



Equate effervescent cold relief tablets (EQ) were used to create training sets and operating procedures to determine the similarity of these tablets as compared to the Alka- Seltzer Plus cold formula (AS). The hygroscopic tablets were sampled out of the wrapper that required the supplementation of fresh samples every half hour. Researchers utilized a great deal of care to acquire spectra from the different areas of each tablet.

Results and Discussion

To validate the robustness of the EQ training set, p-values that are set well above the assigned confidence level of 0.05 will indicate that the sample spectra are between an acceptable level of variance as compared to the spectra of the representative samples included in the training set.

Table 1. Verification of the EQ training set

p-value Result
0.146 PASS
0.204 PASS
0.712 PASS
0.648 PASS


The Mira P is a good technique to verifies that both the EQ and AS tablets are not chemically equivalent, as the p-values in this experiment demonstrated. An additional experiment in which the AS tablets were sampled with the ORS ON and OFF demonstrated the effect of inhomogeneity on verification.

When an inhomogeneous sample is tested with the raster off, each scan of the surface will verify only the components present in a very discreet area. The spectra will therefore vary according to any changes that are present within the chemical composition of the tablet. Sampling with the raster placed in the ON position results in a failed verification test as indicated very low p-values, but it often passed with the raster OFF. If the ingredients within the tablet were found to be especially different in identity, amount and its relative distribution, all scans would be expected to fail.

Table 2. Comparison of AS with EQ training set

p-value ORS ON Result p-value ORS OFF Result
0.013 FAIL 0.533 PASS
0.008 FAIL 0.573 PASS
0.008 FAIL 0.197 PASS
0.180 PASS 0.010 FAIL
0.020 FAIL 0.010 FAIL
0.000 FAIL 0.056 PASS
0.000 FAIL 0.131 PASS
0.000 FAIL 0.082 PASS
0.000 FAIL 0.007 FAIL


The results in the third and fourth columns of Table 2, combined with close comparison of the spectral peaks seen in Figure 1, suggest that the ingredients in each brand of OTC cold tablets are similar in terms of their quantitative analysis. It is important to note that the actual distribution of the active ingredients was found to be different between the brands, thereby resulting in failed verification. This demonstrates the contribution that the Mira P ORS technique makes to sample verification.

Raman spectra of EQ and AS samples

Figure 1. Raman spectra of EQ and AS samples


Verification with p-values by the use of a Mira P handheld Raman spectrometer has been shown to be offer a rapid and convenient method in identifying the content and proportion of active ingredients in two OTC medications that are fairly similar, but not identical. The Mira P has also been shown to sensitive enough to detect any differences in the homogeneity that exists between the brands.

This information has been sourced, reviewed and adapted from materials provided by Metrohm AG.

For more information on this source, please visit Metrohm AG.


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