Hello, I used triples a lot to quantify small molecules in a variety of matrices, and almost always used only one transition to quantify and did not use a confirmation ion.  As long as you have good linearity, solid QCs and a consistent internal standard response, one quantifier is fine.  In fact I've submitted data to regulatory agencies that only has one MRM.  There are plenty of examples in the literature of people validating and using LC-MS/MS methods on one transition:

http://www.sciencedirect.com/science/article/pii/S2095177913000415

A validated LC–MS/MS method for the determination of tolterodine and its metabolite in rat plasma and application to pharmacokinetic study

Journal of Pharmaceutical Analysis

Volume 3, Issue 6, December 2013, Pages 489–499

http://www.malariajournal.com/content/13/1/42

The development and validation of an LC-MS/MS method for the determination of a new anti-malarial compound (TK900D) in human whole blood and its application to pharmacokinetic studies in mice

Malaria Journal 2014, 13:42

http://chromsci.oxfordjournals.org/content/50/3/277.full

Validated LC–MS-MS Method for the Determination of Quetiapine in Human Plasma: Application to a Pharmacokinetic Study

J Chromatogr Sci (2012) 50 (3): 277-282. doi: 10.1093/chromsci/bms001

And here is a paper where they validated a method using flurbiprofen, using one transition:

http://www.scribd.com/doc/209209231/Simultaneous-LC-MS-MS-quantification#scribd

Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

Bioanalysis. 2014 Jan;6(2):151-64. doi: 10.4155/bio.13.289.

If they're worried that you might be measuring a metabolite, the retention time consistency should take care of that since metabolites will elute earlier in RPLC.  If you get no signal in your matrix blank at that RT I would not be worried about inaccuracy.

Having said that, I see confirmation transitions being used more and more, so in the future I would include a second MRM if you get a good signal for the second MRM.

Good luck,

Chris

P.S. I usually monitor three transitions when doing LC-MS/MS, but will only look at one (the others were there as insurance in case that main transition saturated).  I recommend this practice, it often saves you from doing reruns.   

I think it will be nicer if you have 2 daughter ions (from one specific precursor). The confirmation of a target compound can be well proved using quantification, qualifier ions and its ratio (according to Commission Decision 2002/657/Ec.) , Yes, many publications used one daughter ion only. As far as you can prove the accuracy and precision are OK,  I think your method is OK.

I also think your method was ok. Your controls were clean, as you said,  and so their concentrations should be below the limit of detection (LOD), which is lower than your demonstrated LLOQ.  Therefore, the controls were free of the test article (and free of interference also). 

I believe the major aim of additional transitions (qualifiers) is to help to identify and confirm suspected interference. With the qualifier ion ratio in the expected range, you will have a stronger argument that your detection is free of interference.  Perhaps the reviewer was concerned about the potential interference.

With additional transitions, the S/N for the quantifier will suffer somewhat though.  But it is often a price well worth paying.

Dear Huanghui,

LC-MS is a nice and also relyable method. It is however cronic on false positives and matix effects. Therefore in serious work all people do two MRM and report the results separately or provide result and ratio.

It is important both transisions are positive (to make plausible you not hunting a complete gost) and both transitions give the same result otherwise the chance is high at least on of the transitions is disturbed is frightenly high.

The above mentioned EC decision is one of the very few EU documents that make sense to me ;-)

It depends on the level of sample pre-treatment you are using. For a selective sample work up (eg SPE with a deuterated Internal Standard) with good chromatography then you can probably get away with a single transition (although a qualifier may be desireable). However, if you are using a simple sample prep procedure (eg protein precipitation and injection) or simple chromatography then the risk of matrix effects and interferences is greatly increased and a qualifier should be thought of as essential. 

Question: Is it necessary to have a qualifier ion for internal standards also? One of the reviewers for our recently submitted paper thinks so.