Improve the Efficiency of Your Impurity Testing: Impurity Solution Mixtures for use in Pharmaceutical Analysis

Nick Hauser, Product Manager Reference Materials

To ensure the safety of pharmaceutical products, active pharmaceutical ingredients (APIs) need to be tested thoroughly. The accurate detection and control of impurities in drug substances and products is an essential element of ICH and GMP requirements. Reference Materials for these impurities are often hard to find or very expensive. In pharmaceutical quality control (QC) laboratories, APIs and related substances are weighed daily or weekly to make multi-analyte stock or working level solutions. This cumbersome approach introduces the potential for inconsistencies and error, especially with difficult to handle materials.

Our new product range of pharmaceutical impurity solutions saves both cost and labor by providing all regulated impurities in one mix. These impurity solutions are manufactured as Certified Reference Materials (CRMs) according to ISO 17034 and ISO/IEC 17025 double accreditation. Accelerated stability studies are conducted under sub-freezer, freezer, refrigerated, room temperature and 40⁰ C conditions over several weeks to determine any degradation or interaction of the individual analytes. If necessary, individual impurities will be removed from the mixtures and manufactured as single component solutions using conditions that will fortify stability. These single component solutions are manufactured at convenient concentrations to easily be combined with the existing mixtures with minimal sample handling. Also, storage in amber ampoules under argon protects the materials from air, light, and changes in concentration.

Another unique aspect of these solutions is that the individual neat materials that are used as starting materials are Secondary Pharmaceutical Reference Standards. These Secondary Standards are manufactured as Certified Reference Materials and made traceable to a corresponding Primary Compendial Standard. The products are designed to be used as stocks and/or working level solutions.

Table 1 shows the composition of the combination of Acetaminophen impurity mix A-134, 4′-Acetoxyacetanilide (Acetaminophen RC A) solution A-135, 4-Aminophenol (Acetaminophen RC K) solution A-136, and 4-Chloroacetanilide (Acetaminophen RC J) solution C-166 comprising the components required for analysis according to the monograph methods. See compound structures in Figure 1.


Table 1. Acetaminophen impurity portfolio (combining Cat. Nos. A-134, A-135, and A-136).

Component USP Related Compound (RC) EP Impurity
Acetaminophen - -
4-Acetoxyacetanilde A H
N-(4-Hydroxy phenyl)propanamide B B
2-Acetamidophenol C A
Acetanilide D D
4-Nitrophenol F F
4-Chloroacetanilide J J
4-Aminophenol - K


Figure 1. Chemical structures of the Acetaminophen impurities

Acetaminophen 4'-Acetoxyacetanilide N-(4-Hydroxyphenyl)
Acetanilide 4-Nitrophenol 4-Aminophenol 4'-Chloroacetanilide


Figure 2 shows an example chromatogram of the Acetaminophen and related compound solutions (structures of components shown in Figure 1) combined as one sample solution solution, as well as two different tablet types (extra and regular strength) being analysed.


Figure 2. HPLC-UV analysis of a blank (1), the combined Acetaminophen impurities (Cat.Nos. A-134, A-135, and A-136) (2), an extra strength over the counter tablet (3), and a regular strength over the counter tablet (4).

Column: Discovery® C8, 25 cm x 4.6 mm I.D., 5 µm (59354-U)
Mobile Phase: [A]: Methanol, Water, glacial acetic acid (50:950:1)
  [B]: Methanol, Water, glacial acetic acid (500:500:1)
Flow Rate: 0.9 mL/min
Column Temp.: 40˚ C
Detector: UV, 254 nm
Injection: 5 µL
Sample Blank – methanol. Mixture of Acetaminophen impurity mix (A-134), 4′-Acetoxyacetanilide (Acetaminophen RC A) solution (A-135), and 4-Aminophenol (Acetaminophen RC K) solution (A-136), 20.8 µg/mL each individual 25mg/mL in methanol.
Time (min) %A %B
  0.0 80   20
  8.0 80   20
53.0   0 100
58.1   0 100
59.0 80   20

HPLC-UV analysis

  HPLC-UV analysis

HPLC-UV analysis

  HPLC-UV analysis