Sgs lab-anomalous-peaks-en-11


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How To Approach Identification of Unknown Peaks During pharmaceutical sample Analyses.

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Sgs lab-anomalous-peaks-en-11

  1. 1. LIFE SCIENCE I TECHNICAL BULLETIN ISSUE N°49 / JULY 2011HOW TO APPROACH THE IDENTIFICATION OF ANOMALOUS PEAKSDURING PHARMACEUTICAL SAMPLE ANALYSESAUTHOR: NICK TOLTL, PH.D., MANAGER, R&D, SGS LIFE SCIENCE SERVICES, CANADAThe sudden appearance of an unknown peak during an HPLC analysis of a pharmaceutical product can be a criticalfinding causing delays and requiring considerable resource (both time and money) to resolve. The immediate needto investigate an unknown peak is a common problem and is generally accompanied with aggressive timelinesand stressful conditions and the issue can be caused by many things which can range from simple sample prepa-ration contamination, all the way to unexpected degradation/stability properties of the product. In any case, it isessential that a rapid and effective investigation take place to quickly determine the identity of the anomalous peakand determine the root cause of its presence in the product.The source of an unknown peak can be peak in order to ascertain the impact this BEFORE ATTEMPTING PEAKattributed to: peak may have on the affected product IDENTIFICATION and ultimately the patient. However,• Laboratory sample contamination determining the identity of the peak can The isolation and identification of an• Instrument related peaks be challenging because: unknown peak is not a trivial exercise• Method resolution (e.g. a vendor and if done incorrectly, can cause more modification to HPLC column pack- 1. The level of the peak is usually small problems. Prior to making the decision ing altering the column performance (0.05 – 0.2% area percent of the to begin the isolation and identification revealing a previously undetected main peak) process, some in-lab investigations entity) 2. There is limited knowledge on the and standard OOS practices should be• Raw material impurities or contami- source of the peak performed. nation 3. Without a structure, spectral proper-• Product or aliquot degradation (a ties such as relative response factor 1. Re-perform the sample preparation material stability issue or reaction are unknown in scrupulously pre-rinsed glassware with another excipient) 4. The peak may not be well resolved to rule out laboratory contamination• Leachables/extractables (from a from other peaks in the analysis 2. Re-analyze the sample on a different manufacturing step or a container instrument closure system) While each investigation is situation spe- 3. Analyze the raw materials used in• Manufacturing process cross-con- cific, these are some general guidelines the manufacturing process individu- tamination and strategies that can be utilized to ally using the drug product impurity• Some other unknown source achieve success in these endeavours. methodology in an attempt to iden- In a typical study, the strategy for tify the source of the peakRegardless, once an impurity analysis on obtaining the necessary data to identify 4. Review historical data to determinea drug product reveals that an anomalous the unknown peak is not “set in stone” . if the unknown entity had beenpeak causing a failure of a specification, While there is certain flexibility to the present (maybe at lower levels) init jeopardizes the release of material into plan, the basic tools necessary to make a prior batchesproduction, or even has the potential to conclusive identification include (but are 5. If cross contamination is suspected,initiate a product recall. not limited to) diode array, LC-MS/MS, review batch and equipment recordsAs a first step, one needs to assess if accurate mass MS, and NMR. to assess potential candidate com-the peak is real and not an artifact. Once ponentsconfirmed, it is critical to determine theidentity and the source of the unknown
  2. 2. LIFE SCIENCE I TECHNICAL BULLETIN 2STAGE 1 – DIODE ARRAY STAGE 3 – LC-MS/MS AND but this exercise is not trivial. A substan-ANALYSIS HIGH RESOLUTION MS ANALY- tial peak collection effort is required to SES collect enough of the unknown peakOnce the decision is made to initiate the to perform an effective NMR analysis.unknown peak investigation, the first Mass spectrometry is a powerful tool On a case by case basis, various peakdata that should be generated in an un- during the investigation of unknown fraction collection strategies can beknown peak investigation is a UV spec- peaks but caution must also be em- used to obtain enough sample for NMR.trum using a diode array detector. This ployed when interpreting the data Unknown peaks (~0.1%) that are wellserves three purposes: 1) the UV spec- generated using this technique. Points resolved from interferences and havetrum can give some critical structural to consider when analyzing the MS data maximized the on-column load of theinformation about the unknown peak, 2) include: material may still require anywhere fromit may also give an indication if the peak 20-40 injections/collections to obtainis related to the parent molecule being 1. The unknown peak being analyzed the necessary material for 1D and 2Danalyzed and 3) it enables peak track- may not be homogenous NMR experimentation. However, in aning capability during situations where 2. The response of the peak in the MS ideal situation, it is beneficial to collectmethod modifications are required. detector may not correlate with the enough material so that a weight can be UV data measured. Once weighed, the extinctionSTAGE 2 - USE OF VOLATILE 3. In cases where the peak is not coefficient of the unknown peak at theBUFFERS TO ACHIEVE AN LC- homogenous, the MS will afford detection wavelength can be determinedMS COMPATIBLE METHOD data consistent with the moiety which enables the appropriate quantita- in the peak that is the most stable tion of the unknown peak in the HPLCIn the event that the method being used ion, and not necessarily the largest impurity analysis.utilizes volatile buffers that are compat- componentible for LC-MS analyses, then method Upon collection, the mobile phase candevelopment is not required and one That being said, MS and MS/MS data be removed using nitrogen flow. Thecan proceed directly to performing combined with structural libraries can use of heat is not recommended as themass spectral analyses. However, many be an effective way of accomplishing chemical stability of the unknown peakmethods in the pharmaceutical industry a structural elucidation of an unknown is not established. Once dry, the sampleuse phosphate buffer systems in HPLC peak. In addition, the determination of can be submitted for NMR phase. Phosphate buffers are not a high resolution MS data may aid in Consultation with an NMR scientist willcompatible with LC-MS analyses so the the investigation. The use of a 10 ppm establish the appropriate NMR strategyfirst step in the unknown peak investiga- tolerance for suggested molecular to pursue. The utilization of 1D and 2Dtion is to substitute a volatile buffer into formula matches may result in multiple techniques combined with spectralthe mobile phase. For low pH situations, hits. Therefore, information such as the prediction software should afford theformic acid or ammonium acetate are presence of heavy atoms is important structure of the unknown peak.suggested as mobile phase modifiers. to help narrow down the search for theAlso, attempt to adjust the pH to match molecular formula of the unknown peak. DISCUSSIONto the original method pH to minimize Generally, at this point, a holistic analysisshifts in retention times. Lastly, avoid considering the UV profile, molecular The point to remember is that eachusing sodium salts, if possible, in either weight of the unknown peak and how situation is unique and will require anthe buffer or pH adjustment process that compares to the main active individualized case-by-case approach toas counter-ion adducts in MS analyses pharmaceutical ingredient or excipients, identifying the unknown peak. However,make data interpretations difficult. Once fragmentation patterns, and any other the general strategy to identifying anthe mobile phase has been adjusted, available information or historical data unknown peak is summarized in thethe analysis needs to be re-run (using should be performed. flowchart (figure 1).diode array detection) to confirm that thechromatographic profile remains rela- In some cases, GC-MS may also prove It is also very advantageous to collecttively unchanged from the original mobile to be a valuable tool but should also be as much available information beforephase composition and the unknown used with caution due to thermal degra- initiating the investigation. Informationpeak remains well resolved from interfer- dation or lack of volatility. such as structure of the parent molecule,ences. The diode array data also ensures excipient formulation, known degradationthat the unknown peak continues to be STAGE 4 – PEAK ISOLATION pathways of the parent, and historicalthe target of the investigation. AND NMR ANALYSIS data (i.e. forced degradation study data) can all aid in the strategy development. In the event that MS data are incon- Also, it may be beneficial to assess any clusive, further investigation will be subtle changes that may have occurred required to identify the unknown peak. including: At this point, it is suggested that NMR be employed to determine the structure
  3. 3. LIFE SCIENCE I TECHNICAL BULLETIN 3• Changes in raw material suppliers the real amount present is known, a plan• Changes in manufacturing location forward can be developed to resolve or process the issues to: accept or reject the batch,• Any deviations or issues during the amend the specification to control the manufacturing process impurity level, and potentially release the• Changes in the container closure product to market. system or supplier CONCLUSIONLastly, the discovery of an unknown peakusually causes a great deal of stress. The The identification of an unknown peakresolution of the unknown peak issue is a valuable capability to have in oneswill be time critical and there will be troubleshooting arsenal. It requires plan-pressure to complete the investigation in ning as well as the assimilation of manyan expedited manner. It is important to pieces of data. The combination of DAD,understand that this type of an investiga- low and high resolution MS, MS/MS, andtion takes time and there are many tech- NMR data will contribute to the success-nical hurdles that may arise during the ful structural elucidation. Each situationcourse of the work. A reasonable time will offer unique challenges and consid-estimate for the full study is anywhere erations, so the overall strategy can notfrom a few days to ~4 weeks. be a “one size fits all” approach. Different investigational tools may be needed andHowever, performing a due diligence of alternate strategies may be mapped out,all the available information prior to start- but in general, using this guidance as aing the work and following the strategy basis for initiating the anomalous peakoutlined in Figure 1, helps to ensure a investigation will afford the frameworksuccessful outcome and efficient utiliza- needed for expeditious success.tion of time and resources. Once theidentification of the peak is obtained andFIGURE 1: FLOW CHART FOR DETERMINING THE STRATEGY OF UNKNOWN PEAK INVESTIGATIONS UNKNOWN PEAK OBSERVED YES ID/SOURCE OF PEAK DETERMINED? PERFORM OOS INVESTIGATIONS NO PEAK COLLECTION YES ID/SOURCE OF PEAK CONCLUDE DETERMINED? INVESTIGATION NO YES DETERMINE ENOUGH MATERIAL EXTINCTION DAD ANALYSIS TO WEIGH? COEFFICIENT NO YES YES METHOD MS NMR COMPATIBLE? NO MAKE COMPATIBLE OTHER AND PERFORM ID/SOURCE OF PEAK NO INVESTIGATION METHOD DETERMINED? REQUIRED DEVELOPMENT PERFORM LC-MS, LC-MS/MS AND HIGH RES MS
  4. 4. LIFE SCIENCE I TECHNICAL BULLETIN 4To receive future articles on current trends and regulatory updates, subscribe to SGS’ Life Science News at INFORMATIONEUROPE ASIA NORTH AMERICABELGIUM INDIA CANADA+32 10 42 11 11 +91 44 2254 2601 + 1 905 364 ca.pharmaqc@sgs.comFRANCE (PARIS) SINGAPORE USA (FAIRFIELD, NJ)+33 1 41 06 95 93 +65 677 53 034 + 1 888 747 us.pharmaqc@sgs.comFRANCE (POITIERS) CHINA USA (LINCOLNSHIRE, IL)+33 (0) 5 49 57 04 04 +86 21 6115 2197 +1 847 821 us.pharmaqc@sgs.comGERMANY (BERLIN) TAIWAN USA (WEST CHESTER, PA)+49 30 3460 7500 +886 2 2299 3279 ext 2500 + 1 610 696 us.pharmaqc@sgs.comGERMANY (FREIBURG)+49 761 6116 7760 WWW.SGS.COM/PHARMAQCde.pharmaqc@sgs.comGERMANY (TAUNUSSTEIN)+49 6128 744 245de.pharmaqc@sgs.comSWITZERLAND (GENEVA)+41 22 794 8374pharmaqc@sgs.comUK (WOKINGHAM)+44 (0) 1189