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We have developed an method to simultaneously determine the concentrations of a compound and its
metabolite. Cross talk calculations were performed and revealed the following results: ULOQ compound
had no cross talk with LLOQ metabolite but ULOQ metabolite crossed talk with LLOQ compound at 25% in
neat solution. And we observed the same phenomenon in plasma as: ULOQ compound had no cross talk
with LLOQ metabolite but ULOQ metabolite crossed talk with LLOQ compound at 25% in plasma.
Now the problem is we are confused that whether the method could be applied to clinical
pharmacokinetic study.
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Please refer to the Bioanalytical Method Validation (BMV) FDA Guidance, i.e.:
194 Selectivity is the ability of an analytical method to differentiate and quantify the analyte
in the
195 presence of other components in the sample. Evidence should be provided that the substance
196 quantified is the intended analyte. Analyses of blank samples of the appropriate biological
197 matrix (plasma, urine, or other matrix) should be obtained from at least six sources. Each
blank
198 sample should be tested for interference, and selectivity should be ensured at the lower
limit of
199 quantification (LLOQ).
200
201 Potential interfering substances in a biological matrix include endogenous matrix components;
202 metabolites; decomposition products; and, in the actual study, concomitant medication and
other
203 xenobiotics. If the method is intended to quantify more than one analyte, each analyte should
be
204 tested to ensure that there is no interference."
And
208 The accuracy of an analytical method describes the closeness of mean test results obtained by
209 the method to the actual value (concentration) of the analyte. Accuracy is determined by
210 replicate analysis of samples containing known amounts of the analyte (i.e., QCs). Accuracy
211 should be measured using a minimum of five determinations per concentration. A minimum of
212 three concentrations in the range of expected study sample concentrations is recommended. The
213 mean value should be within 15% of the nominal value except at LLOQ, where it should not
214 deviate by more than 20%. The deviation of the mean from the nominal value serves as the
215 measure of accuracy."
So the question is what interferes with what assay and how much within these constraints.
Christopher J. Kemper, Ph.D.
Pharma Navigators, LLC
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It may help to elaborate what you mean by cross-talk. Also, are the compounds chromatographically
separated?
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Thank you for your reply, according to the guidelines, the interference must be less than 20%LLOQ?
can I reduce the ULOQ of analytes？
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The RT of analyte and its metabolite are 1.9 and 2.4 min.ULOQ metabolite had about 25% LLOQ analyte
response at analyte MRM channel.
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As the %interference is greater than 25%, you can not apply this method to clinical pharmacokinetic
study if this intended for regulatory submission. As per USFDA guideline, cross selectivity is a
mandatory experiment for simultaneous quantification methods. Ideally %interference from
drug/metabolite at ULOQ level should not be greater than 20% of metabolite/drug LLOQ.
Regards,
Pavan Kumar Prathipati, M. Pharm, PhD
Research Associate,
School of Pharmacy,
Creighton University,
Omaha, NE
USA
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Thank you for your reply.
Whether this standard is too stringent? At the lowest point of concentration, analyte-containing
metabolites is very low and does not affect LLOQ analyte standard curve, the highest concentration
point metabolites containing the analyte 20% LLOQ, it will not affect the maximum concentration of
the analyte. The highest concentration of metabolites and the lowest concentration of analyte
generally not mix.
Best regards!
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You can reduce ULOQ in order to lower interference. But I don't think with this you will be
completely out of issue. You might face high %RE at LLQC level. Consider looking into following
possible root causes:
1.Inter conversion of metabolite to drug in source.
2. Try selecting a different ion for drug.
Regards
Pavan
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Please explain what you mean by cross talk?
Kind regards,
Bert Meijer
Manager Bio-Pharmaceutical Research Laboratory
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When I hear "crosstalk,” my first thought goes to mass spectrometer related crosstalk. When
injecting only the metabolite in neat solvent, if you monitor only the MRM for the parent, do you
see a peak? If yes, then it is not mass spec related cross talk (which is less common to be anyways
these days).
If there is a peak in the parent channel, at what retention time does it elute, RT of parent, or RT
of metabolite? If it is at RT of metabolite, then there is in-source fragmentation. In that case,
you don’t have to worry too much about it, as long as the %CV of metabolite response from multiple
injections is acceptable. If the RT is the same as the RT for parent, then parent is present in the
solution injected.
If parent is in the solution, the source could be an impurity in the original metabolite powder, or
the metabolite is unstable in the solvent (or mixture of solvents) used. If the impurity is in the
stock, then you need to determine the level of impurity relative to the concentration of parent that
you plan to spike in the same STD or QC. You will probably not spike the ULOQ of metabolite
together with the LLOQ of parent, so you don’t need to compare them.
Let’s assume that you have chosen a 1000 fold range for both parent and metabolite. Also, let’s
assume that the response is linear (which is rarely the case in MS, but just for the exercise). 25%
compared to LLOQ would translate in 0.025% compared to ULOQ. This means, when you mix the parent
and the metabolite in the same standard, or QC, the metabolite will add 0.025% to the parent. This
level is very low and it is defendable that the contribution does not have an impact on the parent
data.
Please keep in mind that these are assumptions, and you would need to measure the actual
contribution. Ideally, the metabolite characterization would indicate the % parent as an impurity,
and that value could be used to evaluate the contribution. Or you can calculate it by UV.
If the metabolite stock is not contaminated with parent, and instead the metabolite is unstable in
solution, you would need to find adequate solvents. Stress test stability in different conditions
and find something that works. Instability is dynamic and it is better to solve the problem, rather
than calculating acceptable levels.
I hope this helps; Good luck!
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Thank you for your reply.If parent is in the original metabolite powder,and metabolite is twice as
parent in human plasma of BE study.Acceptance level can be defined as: Parent in ULOQ metabolite
neat solvent NMT 5% HQC of parent neat solvent？
Regards!
Jia
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If your standard curve range for analyte and metabolite are same, %RE for CC and QC will not suffer.
But if your metabolite CC is X fold as that of analyte, you need to think about it. You are right
"highest concentration of metabolites and the lowest concentration of analyte generally not mix" in
case your standard curve range for analyte and metabolite are same. I guess it would be difficult to
anticipate unknown sample concentrations of metabolite and analyte and its contribution. If you come
across a unknown sample concentration for metabolite at ULOQ level, analyte concentration is
jeopardized.
Regards,
Pavan Kumar Prathipati, M. Pharm, (PhD)
Research Associate,
School of Pharmacy,
Creighton University,
Omaha, NE
USA
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ULOQ metabolite had about 25% LLOQ analyte response at analyte MRM channel(RT analyte). May be
parent is in the original metabolite powder.
I'm sorry I did not describe clearly.
xiej.-a-.kelun.com
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Hi,
Is it possible that your metabolite is not so pure which has your compound. You just need to lower
your ULOQ a bit for your metabolite.
Xiaodong Shen
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Dear,
I would suggest you to estimate analyte and metabolite by two different method. Where you need not
spike the both of the compounds in CC/QC samples. OR find out other source for pure compound if
commercially available.
Regards,
Dipak S Haravani
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