Troubleshooting Method Development – a case study. Diclofenac in milk. Helen Cantwell

Contents Introduction Introduction to the work The Problem The Investigation The Outcome

Introduction Helen Cantwell The State Laboratory, Veterinary Toxicology Residues of veterinary drugs in foods of animal origin. Governed by EU legislation. All methods are LC-MS/MS based. Method development and validation.

Introduction to the work Diclofenac Non steroidal anti-inflammatory drug Used in humans and animals Used topically and orally GI bleeding, ulcers, aplastic anaemia EU: monitor residues in food of animal origin Determination of NSAIDs in milk by LC-MS/MS Diclofenac – no Maximum Residue Limit (MRL) MRPL – 5 µg/kg State Lab report from 2.5 µg/kg Method validated and accredited

The Problem Change in legislation Check existing method Introduction of MRL for diclofenac – 0.1 µg/kg Need to be able to determine down to 0.05 µg/kg Check existing method Transition 1: 294 > 250 Transition 2: 294 >161

The Problem Diclofenac – 294 > 250 Diclofenac at 5ppb

The Problem Diclofenac at 5ppb Diclofenac – 294 > 250

The Problem Diclofenac at 5ppb This is the blank.

The Problem Peak found in the blank. Level will affect determination at 0.05/0.1 ppb. Found regularly. Is it diclofenac? Same retention time. Are there low levels of diclofenac in most milk samples? Is there contamination? Is it an interfering peak?

The Problem Help! Their response: Other labs throughout Europe. European Reference Lab. Their response:

The Problem Help! Their response: It’s diclofenac. Other labs throughout Europe. National Reference Lab. Their response: It’s diclofenac.

The Problem Help! Their response: It’s diclofenac. Other labs throughout Europe. National Reference Lab. Their response: It’s diclofenac. It’s an interfering peak.

The Problem Help! Their response: It’s diclofenac. Other labs throughout Europe. National Reference Lab. Their response: It’s diclofenac. It’s an interfering peak. What peak?

The Problem My involvement begins We need a method to determine diclofenac in milk down to 0.05 ppb.

The Problem My involvement begins We need a method to determine diclofenac in milk down to 0.05 ppb.

The Investigation - Where is it? Analysis of diclofenac in milk requires extracted, matrix calibrants – too many variables. Start with solvent. Carry out multiple injections. Injection solvent (90:10 Water:ACN) Water Air Different vial No septum

The Investigation - Where is it? Peak seen after all injections. Area of peak for all injections is conserved – except for the first injection of a sequence. The first injection has a higher area. Conclusion: The peak is not due to injection onto the system. It is building up in the system until the gradient washes it along the column.

The Investigation - Where is it? To demonstrate this: Carry out multiple blank injections. Injection at time 0. Delay of 10 minutes after the first injection. Delay of 20 minutes after the second injection. Delay of 40 minutes after the third injection.

The Investigation - Where is it?

The Investigation - Where is it? Theory: The peak is not due to injection onto the system. It is building up in the system until the gradient washes it along the column. Outcome: the longer the delay, the larger the peak. Theory confirmed 

The Investigation – Where is it? Theory: The peak is not due to injection onto the system. It is building up in the system until the gradient washes it along the column. Outcome: the longer the delay, the larger the peak. We still don’t know if the peak is due to diclofenac or an interference.

The Investigation – What is it? Structure of Diclofenac

The Investigation – What is it? Structure of Diclofenac Two chlorine atoms Chlorine has 2 isotopes: 35Cl - ~75% 37Cl - ~25%

The Investigation – What is it? Chlorine has two isotopes. A compound with one Cl will have two peaks. A compound with two Cl will have three peaks. A compound with three Cl will have four peaks. 35Cl 37Cl 35Cl2 35Cl37Cl 37Cl2

The Investigation – What is it? Is this distinctive pattern present? Peak height of the three peaks: 9:6:1 Monitoring area but there should still be a repeatable ratio. Monitor M+, M+2 and M+4 Inject diclofenac at 5 ppb to ensure the effect of the peak being investigated is minimal. Carry out blank injections.

The Investigation – What is it? Diclofenac @ 5 ppb Peaks seen for M+, M+2 and M+4 Ratio of the peak areas - repeatable 11 : 7.5 : 1

The Investigation – What is it? Blank injections Peaks seen for M+, M+2 M+, M+2 peaks are very small – maybe not sensitive enough to see the M+4 peak. Compare ratio of M+ and M+2 peak areas. Diclofenac @ 5 ppb M+ : M+2 is 1.5 : 1 Blank Injections M+ : M+2 is 1 : ~1.6

The Investigation – What is it? Ratio of M+ : M+2 for diclofenac and the unknown peak are NOT the same. Unknown peak is NOT diclofenac.

The Investigation – What is it? Confirmation: look at peak maxima. 294 > 250

The Investigation – What is it? Infusion Multiple injections monitoring slightly different ions.

The Investigation – What is it? Confirmation – the unknown peak is not diclofenac. Likely – the interfering peak is due to Triton-X, a component in detergents.

The Investigation – What now? What do I do now? Can I remove the interference? Water – LC-MS/MS, HPLC, Elga Solvents – LC-MS/MS, HPLC, different lots. Interfering peak still present.

The Investigation – What now? Can I selectively tune for diclofenac? Cone voltage Collision energy Capillary voltage Declustering potential Cell exit potential The optimum parameters for the interfering peak ≈ diclofenac. Not possible to selectively tune for diclofenac.

The Investigation – What now? Are there other daughter ions I can use? Literature Fragment diclofenac and look for any other daughters Negative ions: 294 > 214 294 > 178 Positive ions: 296 > 278 300 > 254

The Investigation – What now? Optimise daughter ions. Cone voltage Collision energy Capillary voltage Declustering potential Cell exit potential Temperatures and gases

The Investigation – What now? Carry out blank injections to check for interfering peaks. Negative ions: 294 > 214 – No peak in the blank. 294 > 178 – Peak in the blank – Not suitable. Positive ions: 296 > 278 – No peak in the blank. 300 > 254 – No peak in the blank.

The Investigation – What now? Inject 0.1 ppb diclofenac to assess sensitivity. 294 > 214 (neg) 296 > 278 (pos) 300 > 254 (pos)

The Investigation – What now? Assess sensitivity in matrix (n=5). Blank Matrix 0.1 ppb diclofenac Peak seen with adequate S/N. Background is so high that no peak is seen. High background. Noisy baseline. Peak in blank. 294 > 214 (neg) 296 > 278 (pos) 300 > 254 (pos)

The Investigation – What now? Only one daughter found to be suitable: 294 > 214. Confirmatory analysis requires one parent and two daughters. Diclofenac has two chlorines. Investigate the transition due to the 37Cl isotope. Monitor ion at 296 > 216.

The Investigation – What now? Spike milk samples at 0.1 ppb. Extract and monitor 294 > 214 and 296 > 216. Blank milk Milk spiked with 0.1 ppb diclofenac 294 > 214 296 > 216

The Investigation – Outcome Sucessfully identified 2 parents with one daughter each – confirmatory analysis criteria satisfied. Word of Warning – Use of labelled internal standards. A deuterium labelled internal standard may interfere with an M+2/M+4 isotope and vice versa.

The Investigation – Outcome Using Diclofenac_D4 as internal standard. IS: 298 > 218 M+ : 294 > 214 M+2 : 296 > 216 M + 4 : 298 > 218 Presence of M+4 peak may cause overestimation of internal standard which will affect quantitation.

The Investigation – Outcome M+2 peak and IS. Possible interference due to Gaussian distribution. Use 13C6 labelled diclofenac (300 > 220) instead. The IS contains chorine too – can use IS M+2. Confirm the transitions you have chosen are suitable: Inject IS only – monitor analyte transitions. Inject analyte only - monitor IS transitions. M+2 peak Overlap IS peak

The Investigation – Outcome The peak seen in the blanks was due to an interference not diclofenac. The interference was ubiquitous and not feasible to remove. Two alternate transitions were found. Allowed determination at the level of interest. Allowed determination using the current extraction method. No interfering peaks in the blanks. The method has been successfully validated.

Acknowledgements Acknowledgements: Ed Malone Tom Harbison Sheila Martin Staff of Veterinary Toxicology.