Geoff Carr is the Director of Analytical Development at Patheon located in Mississauga, Ontario. His email is geoff.carr@patheon.com.

Geoff Carr

Visual appearance of pharmaceuticals is important in determining how closely patients comply with their dosage requirements. One problem that is often seen in  tablets is discoloration, or black specks, on the surface. 

	These specks can form because of contaminants in raw materials or from the tablet manufacturing process, and various interactions that may occur during storage. The development of tablet discoloration during storage is often complicated to investigate, because it could involve a variety of interactions between active ingredients, excipients, and packaging materials. This technical case study describes how black specks in tablets were investigated during a stability study. 


	Materials. The pharmaceutical tablets used in this study were film-coated, controlled-release tablets manufactured at Patheon. All excipients used to manufacture the tablets were commercially available and met the 

) standards. Non-active ingredients included:

		The content of API was low, at either 0.1 or 0.4 mg per tablet.

. The microbial limit test was performed using 

 <61> and <62>. Visual inspection was performed on the core, coated tablets, and excipient/active binary mixtures after storage at 40 °C/75% relative humidity (RH) and 60 °C/ambient humidity for 4-6 weeks. Backscattered electron microscopic imaging and elemental composition analysis for the tablets with discoloration were performed using a JEOL JSM 6400 Scanning Electron Microscope (SEM) coupled with Energy Dispersive X-ray Spectroscopy (iXRF EDS/XRF). Mass spectra of SLS were obtained by direct infusion of aqueous solutions of SLS samples into an AB Sciex 3200 LC/MS/MS system with electrospray ionization in both positive and negative modes. Other SLS tests were performed as per the 


	Tablet samples were subjected to a stability study under real time (25 ËC/60% RH) and accelerated (40ËC/75% RH) conditions. After three months, the samples held under accelerated conditions started to exhibit black specks as demonstrated in 

. Because no observations of black specks were reported when these tablets were initially tested for release, after one month at 40 Ë C/75% RH, or following storage at 25 ËC/60% RH, it could be concluded that the specks did not result directly from contamination of tablet ingredients and were not introduced during the manufacturing processes. Instead, they were more likely due to some changes that occurred within the product during the three months when the product was exposed to accelerated storage conditions. An investigation was started to identify a root cause for these observations.

. Microbial contamination was investigated as a potential cause of tablet discoloration, because the warm, moist conditions within a 40 °C/75% RH stability chamber presented a good environment for accelerating growth of microbes.

	Microbial limit tests (MLT) were performed on those tablets whose surfaces showed significant discoloration. Results met all acceptance criteria for total aerobic microbial count, yeasts, molds, and indicator organisms, which ruled out microbial contamination as a root cause of the black specks.

 Because this phenomenon was clearly due to product changes over time, and not some initial contamination, some form of interaction between tablet ingredients was strongly suspected. To study this further, the authors decided to simplify the system by working with binary mixtures. Thus, excipient compatibility tests were performed by compressing binary mixtures (excipient/excipient, excipient/active) of each component of the tablet formulation into tablets and stressing the tablets under 60 °C and 40 °C for up to six weeks. Three different lots of each excipient were tested to evaluate batch-to-batch variability. The core and coated drug product tablets were also stressed to evaluate the compatibility of the coating material with the formulation core. Binary mixture stress results showed that discoloration developed mainly in those samples that contained material from one specific lot of SLS. Further, the extent of discoloration increased with increasing temperature.

	In addition, discoloration shown on both core and coated tablets indicated that it was not caused by incompatibility between the formulation core and any coating material ingredients. 

Imaging and elemental analysis by SEM/EDS

. Back-scattered electron imaging and elemental composition analysis on the areas of discoloration of the tablets were performed by SEM/EDS. Results are shown in 

; the image is in negative mode so that the white area is actually the black speck. As shown in the figure, discoloured areas on the tablets are enriched with the elements sulfur (S) and sodium (Na). The only excipient in the formulation that contained S and Na was the SLS, which further supported a strong relationship between SLS and tablet discoloration.

. Four samples of two lots of SLS were used in this study. Lot A was the same lot used to manufacture tablets that showed discoloration after holding for three months under accelerated conditions. Lot B was a fresh sample from a different supplier. Both lots were held at two different conditions: ambient (as a control) and 60 °C for four weeks. The physical appearance results are summarized in 

	Results indicated that discoloration appeared in stressed samples of SLS from Lot A and did not show up in stressed samples from Lot B or the control samples of either lot. A new batch of tablets was manufactured using the SLS from Lot B and then stressed at 60° C/ambient RH for four weeks. No tablet discoloration was reported over this period. Because both lots of SLS were of a pharmaceutical grade that met the requirements of USP/EP/JP, some further investigation was then conducted to try to establish why the different lots performed differently in tablets.

. Mass spectra of SLS from Lot A and Lot B were obtained by direct infusion of aqueous solutions of each lot into an AB Sciex 3200 MS/MS system. Both positive and negative turbo ion-spray modes were used to collect the MS data. 

 displays the mass spectra of negative mode. Peaks at m/z at 60.0 and 212.0 are from blank, and the peak at m/z 265 corresponds to SLS (C12H25SO4). Apart from these peaks, there were two major peaks (m/z 293 and m/z 97) observed only in the SLS sample from Lot A. These two peaks correspond to the C14 tetradecyl sulfate homologue (C14H29SO4) and residual sulfuric acid (HSO4), respectively.

	The mass spectra of SLS collected from positive mode were crowded with peaks, which made it difficult to interpret. But, overall, the number of peaks from Lot A was much greater than the number of peaks from Lot B. 

	The combined negative and positive mode results indicated that SLS from Lot B has a higher degree of purity than SLS from Lot A, but, more specifically, Lot A showed a significant m/z peak that corresponded to sulfuric acid, which was much less intense in the spectrum of material from Lot B. 

 summarizes the quality control (QC) release test results of the two lots of SLS used in this study. Results showed that, although both lots satisfied the specification requirements of USP/EP/JP, Lot A had a higher level of unreacted alcohols and a lower assay value than Lot B. In addition, the alkalinity and pH value of Lot A are lower than those from Lot B, which again supports the view that this particular lot does contain an excess of sulfuric acid.

. The investigation data demonstrated that the discoloration of the tablets is closely related to the impurities of the SLS used in the tablet formulation. 

	SLS is commercially manufactured by sulfation of lauryl alcohol, followed by neutralization with sodium carbonate. The three most common industrial synthetic processes are:

	For the starting material, lauryl alcohol is usually made from natural products (e.g., coconut or palm kernel oil) followed by a series of hydrolysis and reduction reactions. Due to the natural origin, the lauryl alcohol used for the starting material often contains a minor quantity of C-10, C-14, and C-16 homologues, and they can be further transferred into the final product. Because of the similar chemical/physical property of those homologues, they are not considered to be impurities of SLS. 

	From the synthetic routes, the most common synthetic impurities of SLS are the unreacted residual alcohols, residual sulfuric acid (resulting from incomplete neutralization and/or inefficient purification), salts (NaCl and/or Na

 after the final neutralization reaction. This is probably why compendial monographs of SLS include specification requirements for alkalinity, sodium chloride, sodium sulfate, and unsulfated alcohols content. 

	The impurity profile and levels in the SLS final product depend on the actual sulfation process used for SLS manufacturing and how well the process is controlled. For example, the oleum process is an equilibrium process, which usually leaves large quantities of sulfuric acid in the final reaction mixture, and the unreacted sulfuric acid must be separated from the reaction matrix before the final neutralization step takes place. Therefore, SLS manufactured from this process usually contains a relatively high level of sulfate. On the other hand, for the SO3/air or the chlorosulfonic acid process, the reaction is stoichiometric and fast. When reaction conditions are tightly controlled, there is much less sulfuric acid or sulfate formed, resulting in a product of higher quality.

	It is possible that during the manufacturing of SLS (Lot A), the neutralization step had not been completed, resulting in the presence of residual sulfuric acid in the final product. This could explain the low alkalinity and pH value reported in Lot A from the QC release test results (Table II). Upon storage at higher temperature, the residual sulfuric acid undergoes dehydration reactions with the carbohydrate excipients (e.g., lactose) in the tablet formulation, resulting in the formation of black specks.


	The most likely mechanism of tablet discolouration was related to the dehydration reaction between the residual sulfuric acid (due to incomplete neutralization during SLS manufacture) and the carbohydrate excipients present in the formulation, upon storage at higher temperatures.

	Tablet discoloration is closely related to the quality of SLS used in tablet formulation, and the quality of SLS depends on the synthetic route and degree of process control used by the SLS during manufacture.
	Current specifications of SLS in compendial monographs are not sufficient to control the quality of SLS, because they do not discriminate between different SLS lots, including those that may cause this type of drug product failures. Therefore, it is recommended to develop a testing requirement in the USP/EP/JP monograph that more tightly controls the formation of residual sulfuric acid and other impurities during SLS manufacturing.


	Submitted: January 5, 2015.
	Accepted: January 26, 2015.

Pharmaceutical Technology Outsourcing Resources Supplement

 When referring to this article, please cite it as G. Carr, "Black Specks in Tablet Stability Samples," 

Articles

A study of root cause in stability samples suggests the need for tighter control of the sodium lauryl sulfate manufacturing processes.

Black Specks in Tablet Stability Samples

Within any pharmaceutical manufacturing facility it is crucial to ensure that following the manufacture of a product, equipment has been thoroughly cleaned to avoid risks of any carry over into the following product. The process by which this is achieved is called cleaning validation, and an approach for conducting such validations is to develop a sequence of operations likely requiring the application of solvents or detergents and rinses. It must be demonstrated that this sequence of operations can remove traces of product residues down to levels that do not present risks to patients that may receive them as carryover components in the next product manufactured on the same equipment train.  To collect data, swab samples taken from equipment surfaces or rinsates (i.e., washes that are used for the rinsing out of equipment after they have been treated with detergent) are analyzed. Typically, samples of the final washes are taken for analysis.  

If a validated cleaning procedure has not been developed, it will be expected that swab or rinsate analyses will be conducted on each occasion to demonstrate effectiveness of the cleaning procedure; this practice is referred to as cleaning verification. 

For both cleaning validations and verifications, analytical testing of samples is a crucial step. Typically, analytical procedures that are specific for the APIs that were used for the manufacturing of previous products are applied. It is expected that a separate analytical procedure will be developed and validated for each individual API that is handled in any manufacturing facility. High-performance liquid chromatography (HPLC) is probably the most widely used analytical procedure for this application with ultra-high performance liquid chromatography (UHPLC) gaining increasing popularity, but liquid chromatography-mass spectrometry (LC-MS) also offers potential opportunities.  

			For mass detection to work, various analyte components that elute from the chromatographic column must become ionized. Ionization may be achieved using electro-spray ionization (ESI). An example of an MS detector (MDS SCIEX, AB Sciex) is depicted in 

 depicts the layout of the electrospray of an MS detector. As shown in 

, the effluent from the HPLC column is directed through a nebulizer that is maintained under a high voltage. The voltage can be selected for either positive or negative polarity depending on the conditions required for the components of interest. The charged droplets are subjected to heat to remove solvent, which creates a charged aerosol that is directed into the MS quadrupole.  

Figure 1: High-performance liquid chromatograph (Agilent 1200) with attached triple quadrupole mass spectrometer (MDS SCIEX). (ALL FIGURES ARE COURTESY OF THE AUTHOR)			

A quadrupole system provides the mechanism for discriminating between different components within the sample under test on the basis of M/z values, in which M is the molecular weight and z is the magnitude of charge. For example, the ESI process could lead to single or double charged species or even higher depending on the chemical structure of the component concerned. By varying the magnetic field within the quadrupole system, species of different M/z values may either be eliminated or directed onto the instrument detector.  

Figure 2: Diagram showing the layout of the electrospray system of a mass-spectrometer detector; LC is liquid chromatograph.			

 is for a single quadrupole instrument (i.e., LC-MS). An even more discriminating system is provided by a triple quadrupole approach (i.e., LC-MS-MS). In this case, the species that are selected by the first quadrupole (Q1) are directed into a second quadrupole (Q2), which serves to collect the particular M/z species of interest and then direct them into a third quadrupole (Q3). Within Q3, the species are subjected to collisions with other atoms or molecules that are often provided by allowing a small volume of nitrogen into the system. Collisions lead to fragmentations and resulting fragment ions may then be used as a basis of the analysis required.   

The price of a typical HPLC system with UV detection is approximately US$77,000. The price of a typical triple quadrupole MS detector is approximately US$193,000, and the user must still also purchase an HPLC front end for the system. In addition, at least two systems are recommended for any organization considering the use of the LC-MS approach so that a back-up system is available. Pharmaceutical manufacturing facilities are scheduled to very tight timelines. Any downtimes, such as those due to cleaning following the manufacture of a batch and analytical testing of swabs and release formalities prior to manufacture of the next product, must be kept to a minimum; delays due to breakdowns of analytical equipment cannot be tolerated.  

. HPLC is most often applied using ultraviolet (UV) light absorption detectors, which require that the chemical structures of analytes of interest include a chromophore (i.e., chemical structural features that absorb UV light). For most compounds of pharmaceutical interest, this is frequently the case, but there are exceptions which may require other types of detection. MS detection is appropriate for any analyte, provided that it can be ionized. Since most can be ionized, this approach has almost universal applicability.    

Analysis for cleaning residuals is an example of an application that requires very high sensitivity. LC-MS is capable of providing reliable quantitation of trace components from 100 to 1000 times lower than HPLC with UV detection. 

HPLC runtimes are extremely variable depending on the particular method concerned. Run time is the time from the injection of the analytical solution into the chromatograph to the end of the resulting chromatogram. Run times of 5–10 or even 20 min. are not unusual. With LC-MS, typical run times are one to two min. System set-up time is also much reduced for LC-MS, as the same LC column and mobile phase can be applied for many applications. Each analysis will involve a sequence of injections, including those required for system suitability, standard solutions, and sample solutions. Chromatograms then need to be processed into analytical reports, and data must be reviewed and receive lab organization approval. Overall, Patheon has found that lab turnaround time for analysis of swabs is about 24 h with HPLC and about 8 h with LC-MS. 

			The author would like to gratefully acknowledge the valuable help that was provided by Heng Yao in the preparation of this article. 

is director of Analytical Development at Patheon, Mississauga, Ontario,  

The author describes how liquid chromatography-mass spectrometry works and explains some of its advantages and disadvantages, particularly compared to high-performance liquid chromatography.