Formaldehyde is a highly reactive impurity that can be found in many pharmaceutical excipients. Trace levels of this impurity may affect drug product stability, safety, efficacy, and performance. A static headspace gas chromatographic method was developed and validated to determine formaldehyde in pharmaceutical excipients after an effective derivatization procedure using acidified ethanol. Diethoxymethane, the derivative of formaldehyde, was then directly analyzed by GC-FID. Despite the simplicity of the developed method, however, it is characterized by its specificity, accuracy, and precision. The limits of detection and quantification of formaldehyde in the samples were of 2.44 and 8.12
Although considered pharmacologically nonactive, pharmaceutical excipients have critical effects on drug product safety, efficacy, and quality. During early stages of drug formulation development, excipients are evaluated for appropriateness by studying their physical and chemical compatibility with drug substances under stressed and accelerated conditions. Depending on the results of the compatibility studies, formulator can exclude incompatible excipients from later stages of formulation development. Chemical incompatibility with drug substances may result not only from direct reaction with excipients but also from reaction with excipient impurities. These impurities include formaldehyde which could be generated by autoxidation degradation of many pharmaceutical excipients such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) [
Pharmacopoeial monographs of pharmaceutical excipients rarely require testing for formaldehyde specifically [
Formaldehyde has a low molecular weight and a high chemical reactivity. Furthermore, it has little UV activity and low detector sensitivity and specificity. Besides, it is soluble in both water and organic solvents. As a consequence, it is difficult to directly extract and determine formaldehyde in a specific, accurate, and sensitive way. Therefore, most of the analytical methods used for formaldehyde analysis employ chemical derivatization to improve its stability and modify the physicochemical properties to enhance its detectability [
Many analytical methods have been developed to determine formaldehyde in pharmaceuticals and cosmetics. These methods are based on colorimetric, spectrophotometric, fluorescent, capillary electrophoresis, HPLC, GC, and GC-MS techniques [
As most of the spectrophotometric methods have some disadvantages, while other chromatographic methods are complex to be performed, the aim of this research is to develop and validate a static headspace (SHS) GC-FID method to determine formaldehyde, and then to use it as a screening method and a quality control tool to analyze this impurity in commonly used pharmaceutical excipients.
High-purity diethoxymethane (≥99.0%) was purchased from Aldrich (USA). Absolute ethanol (99.9%) and pure formic acid were purchased from Panreac (Spain). Formaldehyde solution (37–41%) was obtained from SCP (England). ACS grade
Experiments were conducted by Agilent Model 7890A gas chromatograph equipped with mass selective detector (MSD model 5975C) and associated with GC sampler 80 enhanced with Agilent PAL headspace option. Agilent MSD Productivity ChemStation E.02.01.1177 software was used to control, acquire, and process the chromatographic data. A 30 m × 0.25 mm i.d. ZB-WAX column with 0.25
The headspace autosampler parameters were set as follows: incubation temperature: 70°C; incubation time: 25 min for PVP samples and 15 min for PEG samples; syringe temperature: 75°C; agitation speed: 500 rpm; syringe injection volume: 800
The injector was maintained at 170°C with a split ratio of 1 : 25. The column oven temperature program involved an initial temperature of 35°C for 5 min and increased at 40°C/min to 220°C and held for 1 min. The carrier gas was helium (99.999%) at a constant flow rate of 0.9 mL/min. FID was set at 280°C for quantification. MS detection was carried out at 230°C with full scan (31–250 amu) for identification.
Samples were prepared by directly weighing 250 mg of the tested excipient into a 20 ml amber headspace vial (Supelco). After that, 5 mL of the solution of 1% (w/w)
The concentration of formaldehyde solution was determined by applying the iodometric method described in British Pharmacopoeia 2013 [
Standard solutions of formaldehyde were prepared in the acidified ethanol (1%
The method was validated in terms of specify, linearity, accuracy, repeatability, intermediate precision, limit of detection, and limit of quantification according to British Pharmacopoeia 2013 [
Results were statistically processed by
Overlaid chromatograms show the conversion of formaldehyde in acidified ethanol to the corresponding derivative. (A) Ethanol; (B) formaldehyde derivative; (C) diethoxymethane standard.
To further confirm the identity of formaldehyde derivative, EI mass spectrum of the corresponding peak (shown in Figure
EI mass spectrum of formaldehyde derivative.
In order to explore the factors that leaded to such low recovery of formaldehyde derivative from PVP K-30, the sample matrix effect and the previous incubation parameters were checked. First, the experiments carried out on the ethanolic solution of diethoxymethane standard showed that the incubation time of 15 min at the temperature of 60°C was sufficient for diethoxymethane to reach the static headspace equilibrium. Second, the mean recovery of diethoxymethane standard was 110% (
Effect of incubation time at 60°C on formaldehyde derivative peak area in PVP K-30 when the sample concentration is 50 mg/mL (blue box) and 100 mg/mL (red box).
As a consequence, sample dilution from 100 mg/mL to 50 mg/mL gave the advantage of lowering the incubation time to 30 min (instead of 45 min at least). Therefore, the sample concentration of 50 mg/mL was selected to complete the other experiments.
Effect of incubation temperature on response (
Incubation temperature (°C) | Response (mean ± SD) |
---|---|
60 | 230299 ± 4872 |
70 | 233366 ± 4779a |
aNo significant difference when compared with incubation at 60°C.
Effect of incubation time at 70°C on formaldehyde derivative peak area. Formaldehyde in PVP K-30 (red box) and formaldehyde in PEG 400 (blue box).
As a consequence, the increasing of incubation temperature from 60°C to 70°C leaded to a decrease in the incubation time from more than 15 min to 15 min for PEG 400 and from 30 min to 25 min for PVP K-30. Therefore, the incubation temperature of 70°C was selected.
Diethoxymethane formation reaction is susceptible to water presence which can reverse the reaction [
Effect of water content on formaldehyde determination (
Percentage of water relative to sample weight (%) | 0 | 5 | 10 | 20 |
---|---|---|---|---|
Formaldehyde concentration ( |
1.45 ± 0.025 | 1.48 ± 0.009a | 1.48 ± 0.025a | 1.48 ± 0.015a |
aNo significant difference when compared with the control (0% water).
As a consequence, excipient samples containing water ≤ 20% (w/w) can be analyzed without any significant effects on formaldehyde determination. Higher amounts of water were not experimented because the acceptable limits of water content stated in the monographs of PEG and PVP are 1–2% and 5%, respectively [
The specificity of the method was determined by comparing the chromatograms (Supplementary Material Figure
The linearity of the method was evaluated from injection of nine concentrations of formaldehyde at the range of 0.25–50
The accuracy of the optimized method was determined by spiking the excipient samples with known amounts of formaldehyde at four concentration levels (
The precision of the method was evaluated in terms of the repeatability (expressed as intraday precision) and the intermediate precision (expressed as interday precision). The repeatability of the method was established from triplicate (
Accuracy, intraday, and interday precision of the developed method.
Formaldehyde spiked ( |
PVP K-30 | PEG 400 | ||||
---|---|---|---|---|---|---|
Mean recovery % ( |
Intraday precision (RSD%) ( |
Interday precision (RSD%) ( |
Mean recovery % ( |
Intraday precision (RSD%) ( |
Interday precision (RSD%) ( | |
10.0 | 104.27 | 2.65 | 1.92 | 87.76 | 0.75 | 3.18 |
50.04 | 98.14 | 1.48 | 1.56 | 89.79 | 1.59 | 3.24 |
500.4 | 98.99 | 2.96 | 3.61 | 97.70 | 2.04 | 2.67 |
1000.9 | 97.89 | 1.52 | 1.56 | 101.12 | 2.07 | 2.05 |
The limits of detection and quantification (LOD and LOQ, resp.) were evaluated based on signal-to-noise ratios of 3 and 10, respectively, and the respective values were found to be 121.80 and 406.00
Changing the flow rate within ±0.1 mL/min had no effect on the area of formaldehyde derivative peak, and all RSDs were less than 5%. This indicates the robustness of the method.
Little analytical methods have been developed to determine formaldehyde in pharmaceutical excipients [
Comparison between the methods used to determine formaldehyde in excipients or drug substances.
Analytical technique | Derivatization and/or extraction times (min) | Run time (min) | Approx. recovery (%) | Approx. precisiona (%) | Approx. LODb (ppm) | Ref. |
---|---|---|---|---|---|---|
GC-FID | 5 | >7 | No data | No data | 7 | [ |
GC-MS | 240 | 15 | 86–99 | 3.7 | 0.02 | [ |
SHS-GC-FID | 30 | 5 | 85–97 | ≤3 | 0.05 | [ |
SHS-GC-MS | 20 | 28 | No data | 3.1 | 0.05 | [ |
SPME-HS-GC-MSc | 60 | 22.5 | 106.5–113.5 | 3.13–13.19 | No data | [ |
HPLC-UV | No data | ≥35 | 84–97 | 0.5–1.2 | 0.5 | [ |
HPLC-UV | 60 | 30 | No data | No data | 0.03 | [ |
HPLC-UV | No data | No data | 94.9–102.9 | No data | 10 | [ |
Proposed method | 15 or 25 | 11 | 87.8–104.3 | 0.75–3.6 | 2.44 | — |
aIncluding inter- and intraday precision; bLOD in sample; cSPME: solid-phase microextraction.
Compared to other methods, the time required to complete formaldehyde determination using the developed method was generally faster. Recovery obtained with the presented procedure was comparable with those reported by others. Precision of the method was also close to those mentioned by others; however, it was better than that for SPME-based method. Although the proposed method had lower sensitivity than most of the other methods, however, it gives the opportunity to detect and determine formaldehyde impurity in pharmaceutical excipients below the limit stated in British pharmacopoeia (15 ppm for free formaldehyde in PEG) [
PVP and PEG are widely used in many pharmaceutical applications and dosage forms. They are utilized in drug products over a wide range of concentrations. These concentrations can reach up to 67% for PEG 400 and 25% for various grades of PVP [
The optimized SHS-GC-FID method was applied to analyze the presence of formaldehyde in several commercial PEG and PVP samples. The results of the analysis are summarized in Table
Formaldehyde level in the excipient samples.
Company | Excipient | Formaldehyde level ( |
---|---|---|
A | PVP K-30 | NDa |
B | PVP K-30 | NDa |
C | PVP K-30 | 3.51b |
D | PVP K-25 | 8.04 |
E | PEG 400 | 22.47 |
F | PEG 400 | 3.64b |
G | PEG 300 | 190.58 |
aNot detected; bformaldehyde level < QL.
Formaldehyde content in the excipient samples was extensively varied from 8.04 to 190.58
Formaldehyde was not detected in PVP K-30 samples from the companies A and B. However, PVP samples (from the companies C and D) contained trace amount of formaldehyde. Actually, there is no specific pharmacopoeial limit for formaldehyde in PVP, but there is a general limit for aldehydes (500 ppm) which expressed as acetaldehyde [
According to British Pharmacopoeia, the limit of formaldehyde in PEG is 15 ppm [
Although PVP K-25 from the companies C and D and PEG 400 from the company F contained trace levels of formaldehyde, however, such levels may cause critical drug products instability and degradation, especially when excipient/active pharmaceutical ingredient ratio is high, according to many literatures [
A SHS-GC-FID method was developed and validated for determination of formaldehyde in pharmaceutical excipients. Samples were simply prepared in headspace vials by adding acidified ethanol as a diluent and derivatization reagent. After that, the vials were automatically moved to the incubator where formaldehyde derivative was simultaneously formed and extracted. The developed method was linear, specific, accurate, and precise over the specified range. The limits of detection and quantification were 2.44 and 8.12
The authors declare that they have no conflicts of interest.
The authors greatfully acknowledge the Assistant Prof. Dr. Manal Daghestani, Dr. Faten Alsaka, Dr. Ghassan Abochameh, and Ph.D. student Yamen Alsalka for helping in carrying out analysis and data processing.
Figure S1: representative GC-FID chromatograms of specificity study.
Figure S2: GC-FID chromatogram of PEG 400 sample spiked with formaldehyde. DEM: diethoxymethane.
Figure S3: GC-FID chromatogram PVP K-30 sample spiked with formaldehyde. DEM: diethoxymethane.