Laboratory needs for analyzing PGIs in API vary. Here we developed both a fast analysis of sulfonate esters on the Rxi-5Sil MS column, and a comprehensive method for sulfonate esters and alkyl halides on the Rtx-200 column.
Compounds that are used in the synthesis of active pharmaceutical ingredients (API), or reaction byproducts that form during synthesis, have the potential to remain as impurities. Some of these compounds are potentially genotoxic impurities (PGI) and may raise concern about cancer and/or birth defects.
Four structural classes of PGIs are discussed in this article. The first three classes, known collectively as sulfonate esters, include mesylates, besylates, and tosylates (Figure 1). These alkylating sulfonic acid esters may form when sulfonic acid reacts with an alcohol solvent. The first three classes are differentiated by the group that forms an ester with the sulfur: mesylates contain a methyl group, besylates contain a phenyl (benzyl) group, and tosylates contain a toluene group. The fourth class of PGIs tested here, alkyl halides, consists of short alkyl chains with halogen constituents. Since alkyl halides are polar and very volatile, they are not retained well on thin film stationary phases. This can make analysis of a mixture of sulfonate esters and alkyl halides quite problematic.
Two options for the analysis of PGIs in API have been developed to meet different laboratory needs. The first option is a fast method for the analysis of sulfonate esters on the Rxi-5Sil MS column. The second option is a comprehensive method for the analysis of both sulfonate esters and alkyl halides on the Rtx-200 column. Both methods require very little sample preparation, which helps increase laboratory productivity.
Option 1: Fast Analysis of Sulfonate Esters
Scientists from Merck, in collaboration with Restek, have developed a fast method for the analysis of sulfonate esters on the Rxi-5Sil MS column. Since PGIs can differ dramatically from one another, several types of API were spiked and analyzed to ensure the robustness of the method. Figure 2 shows an example of API spiked at 1 ppm, which is the threshold for toxological concern (TTC) for PGIs in API as set by the EMEA. Depending on the dose of API to the patient, it may be necessary to detect levels of impurities as low as 1 ppm in order to meet requirements. A linearity study was also performed and shows that this method is linear for sample concentrations from 1 ppm to 1,000 ppm in API (Figure 3).
The use of a thin film Rxi-5Sil MS column allows for fast analysis of active PGI compounds. Since the Rxi-5Sil MS column is very selective toward sulfonate esters, a fast oven program can be used to speed analysis. This method allows for the analysis of selected sulfonate esters in less than 4.5 minutes. However, note that since the Rxi-5Sil MS column is a mid-polarity stationary phase, the use of polar sample solvents, such as methanol, is not recommended. Due to the polarity of this phase, splitless injections of sample solvents with water may cause peaks to split. The method shown here utilizes a 10:1 split, which allows for the injection of relatively polar sample solvents, such as 90:10 acetonitrile:water.
Figure 2: Fast separation of sulfonate ester PGIs in API (1 ppm).
|Column||Rxi-5Sil MS, 20 m, 0.18 mm ID, 0.18 µm (cat.# 43602)|
|Inj. Vol.:||1.0 µL split (split ratio 10:1)|
|Liner:||Gooseneck Splitless (4mm) w/Wool (cat.# 22405)|
|Inj. Temp.:||250 °C|
|Oven Temp.:||50 °C (hold 1 min) to 150 °C at 20 °C/min to 250 °C at 50 °C/min|
|Carrier Gas||He, constant pressure (10.19 psi, 70.3 kPa)|
|SIM Program:||80, 109, 123 m/z|
|Transfer Line Temp.:||280 °C|
|Acknowledgement||Merck & Co., Inc|
Option 2: Comprehensive Method for Analysis of PGIs
Although the thin film Rxi-5Sil MS column allows for fast analysis of sulfonate esters, the smaller, more polar alkyl halides are not well retained. To take advantage of the halogen constituents on the alkyl halides, a thick film Rtx-200 column was used to develop this comprehensive method for both volatile alkyl halides and less volatile sulfonate esters. Since the Rtx-200 column has a fluorinated stationary phase, the alkyl halides are well-retained (Figure 4). Note that all of the alkyl halides elute at a low temperature and some of the more volatile compounds elute prior to the sample solvent (acetonitrile). Because of this, the solvent cut time must be carefully measured. The Rtx-200 column is also selective for sulfonate esters, providing baseline resolution for 20 out of 22 of the compounds analyzed. Additionally, the increased polarity of the fluorinated Rtx-200 phase allows for the use of splitless injection of more polar sample solvents, such as methanol.
If the analysis of alkyl halides is not a laboratory concern, a thin film Rtx-200 column may be used for faster analysis of sulfonate esters. A linearity study was conducted to ensure that this method is linear over the expected range of sulfonate esters in API. Samples were prepared at 8 concentrations ranging from 10 ng/mL to 10,000 ng/mL in 90:10 ACN:H2O. This is equivalent to 1-1,000 ppm in API for samples prepared at 10 mg of API/mL of sample solvent. This method shows acceptable linearity for all 15 sulfonate esters analyzed (Table I).
Table I: Excellent linearity is also obtained using the comprehensive PGI methodology.
|Compound Name||R-squared Value|
|dimethyl sulfate (DMS)||0.9984|
|methyl methanesulfonate (MMS)||0.9988|
|ethyl methanesulfonte (EMS)||0.9988|
|isopropyl methanesulfonate (iPMS)||0.999|
|diethyl sulfate (DES)||0.999|
|diisopropyl sulfate (DPS)||0.9993|
|di-n-butyl sulfate (DBS)||0.9996|
|methylbenzene sulfonate (MBS)||0.9991|
|benzenesulfonic acid ethyl ester (EBS)||0.9995|
|p-toluenesulfonic acid ethyl ester (ETS)||0.9996|
|p-toluenesulfonic acid n-propyl ester (nPTS)||0.9994|
|benzenesulfonic acid n-butyl ester (nBBS)||0.9995|
|isopropyl p-toluenesulfonate (iPTS)||0.9996|
|p-toluenesulfonic acid n-butyl ester (nBTS)||0.9994|
|8-point standard curves, 1-1,000 ng/mL (equivalent to 1-1,000 ppm in API matrix)|
Figure 4: Small, polar alkyl halides are well-retained on the fluorinated Rtx-200 column, as are less volatile sulfonate esters.
|12.||di-Isopropyl sulfate (DPS)|
|13.||di-n-Propyl sulfate (DNS)|
|14.||Dibutyl sulfate (DBS)|
|15.||Methylbenzene sulfonate (MBS)|
|16.||Ethylbenzene sulfonate (EBS)|
|17.||Methyl toluenesulfonate (MTS)|
|18.||Ethyl toluenesulfonate (ETS)|
|19.||n-Butyl benzenesulfonate (nBBS)|
|20.||n-Propyl toluenesulfonate (nPTS)|
|21.||Isopropyl toluenesulfonate (iPTS)|
|22.||n-Butyl toluenesulfonate (nBTS)|
|Column||Rtx-200, 30 m, 0.25 mm ID, 1.0 µm (cat.# 15053)|
|Inj. Vol.:||1 µL split (split ratio 10:1)|
|Liner:||4mm Gooseneck Splitless w/Wool (cat.# 22405)|
|Inj. Temp.:||220 °C|
|Oven Temp.:||40 °C (hold 8.3 min) to 70 °C at 70 °C/min to 115 °C at 40 °C/min to 250 °C at 30 °C/min to 300 °C at 15 °C/min (hold 3 min)|
|Carrier Gas||He, constant flow|
|Flow Rate:||1 mL/min|
|Transfer Line Temp.:||280 °C|
|Scan Range:||20-250 amu|
Minimal sample preparation is needed to successfully run these methods. Samples of neat API were simply diluted to a concentration of 10 mg/mL and injected into the GC. However, care must be taken with sample solvent selection, since many APIs are in salt forms and cannot be dissolved in 100% organic solvent. To aid dissolution, up to 10% water may be mixed with a water-miscible organic solvent. The sample solvent used in this application was 90:10 acetonitrile:water.
Since samples are prepared at a relatively high concentration, nonvolatile API may build up in the liner and/or on the head of the column after repeated injections. Care should be taken to regularly change liners and seals to avoid problems with chromatography stemming from contaminated parts.
Some PGIs, such as isopropyl benzene sulfonate, break down in both the inlet and column. These breakdown products may persist in the analytical column, causing an elevated baseline that resembles a bleed profile. If isopropyl benzene sulfonate is present in the sample, a longer bake-out time (10-15 min. @ 300 °C) is recommended at the end of each analytical run in order to remove any degradation products present on the column.
Care must be taken when choosing SIM groups for PGI compounds during method development. Because these analytes are very similar to one another, many of them share common ions. It is good practice to pick two or three ions to monitor for each compound and to use relatively unique ions for each PGI to aid in peak identification.
Since potential genotoxic impurities are of increasing concern for both regulatory bodies and consumers, the importance of effective methods for detection and quantitation of these compounds is growing. As a result of collaboration between Merck and Restek, two easy, sensitive options are now available for the analysis of PGIs in API using inert, selective columns from Restek.