1. How do I measure pump flow accuracy?



2. What is the shelf life and expiration date of the solutions in the HSQ Kit™?



3. Can I measure the extra column volume of my system and compare it to extra column volume of another instrument?



4. Why are there six linearity solutions? Do I need all of them?



5. What are the linearity solutions?



6. What are the differences between the linearity solutions?



7. What is the Gradient Visualization Solution?



8. Why do the solutions say Chemical Solutions on them?



9. Which solution do I use for Injector Carry Over Testing?



10. How do I determine wavelength accuracy below 230nm?



11. Can I get MSDS (Material Safety Data Sheets) for these solutions?



12. Can this kit be used to measure the LOQ and LOD differences between instruments in my lab?



13. How many injections should I expect on the HSQ Kit HPLC Column?



14. What is the acceptable wavelength band width for HPLC detectors?



15. I currently use a glass filter, why should I use these solutions instead?



16. Will the FDA accept the Excel Software in this kit as valid?



17. How do I store the HSQ Validation HPLC Column in the HSQ Kit?



18. How many tests will I get out of one kit?



19. What is the mobile phase used in the HSQ Kit and how much is used?



20. Do the different tests in the kit use the same method conditions?



21. Are column oven temperatures qualified by the HSQ Kit?



22. What is the operating range for temperature with the HSQ Kit?



23. Having difficulties with wavelength accuracy readings.



24. In my older HSQ Kit, the tailing factor minimum was between 0.9 and 1.1, in the latest version, there is no minimum requirement. How does this effect my SOP written with the older kit and is the newer version just as valid and accurate?



25. What is the difference between Verification, Validation and Qualification?



26. I am getting a different retention time for the peaks in the Resolution Test Mixture on different systems. What impact does this have on the integrity of my data?



27. What is the effect of extra column volume for different HPLC instruments on the same method?



28. What are the peaks/analytes in the Resolution Test Mixture?



29. What is the difference between the new HSQ Kit™ and older PQ Kit™?



30. I have my SOP written with the older PQ Kit™ using the longer column. Will you still sell it and support it?



31. Can the L3 linearity solution be used for wavelength accuracy?



32. The instructions which are given in the PDF file and on the Excel sheets are very detailed and a bit overwhelming from my perspective. What should I do? How can you help?

1. How do I measure pump flow accuracy? [top]
Use a dry 5ml volumetric flask and a calibrated stopwatch to confirm the flow accuracy at 1.0 mL/minute. Note that this flow rate check is not meant to replace the typically more extensive Operational Qualification checks at several flow rates. Rather it is to document the suitability of the pump to perform various PQ Test protocols described in this kit.

2. What is the shelf life and expiration date of the solutions in the HSQ Kit™? [top]
Shelf life and stability data has been collected for 18 months with no observed instability. The expiration date is set for 18 months but the shelf life is longer.

3. Can I measure the extra column volume of my system and compare it to extra column volume of another instrument? [top]
Yes, that is one of the added features of the MicroSolv HSQ Kit™. Using the Rs Test Mixture, and the Excel template, using sophisticated algorithms, a very good estimation of your instruments extra column volume is easily determined. To compare it to another instrument, you must use the same column under the exact same conditions and run the same test. It is very valuable information for labs that transfer or move methods from one instrument to another.

4. Why are there six linearity solutions? Do I need all of them? [top]
The MicroSolv HSQ Kit™ is supplied with six linearity solutions to offer a very wide range of concentrations but to be PQ compliant, you may need less depending on your internal SOP.

5. What are the linearity solutions? [top]
The HSQ Kit’s linearity solutions are made under GMP conditions with calibrated balances and following a strict protocol and comprise Caffeine dissolved in 90:10 Acetonitrile:0.1% Acetic Acid.

6. What are the differences between the linearity solutions? [top]
They are all different, precise concentrations of Caffeine that allow you to easily and accurately determine the linearity of your detector. L3 solution is the only linearity solution that has a Uracil peak present. This peak is used as a pump flow stability marker and has no effect on caffeine or linearity tests.

7. What is the Gradient Visualization Solution? [top]
The HSQ Kit’s Gradient Visualization Solution is made under GMP conditions with calibrated balances and following a strict protocol and comprise 1.0 ml/mL Uracil and caffeine in 10% Acetonitrile/water with 0.1% Acetic Acid.

8. Why do the solutions say Chemical Solutions on them? [top]
Chemical Solutions is the brand name of the these solutions. They are made in a GMP facility that is certified by the FDA.

9. Which solution do I use for Injector Carry Over Testing? [top]
You should use the L6 solution which is the highest concentration of Caffeine, 0.3490 mg/mL. This solution is most likely to cause carry over.

10. How do I determine wavelength accuracy below 230nm? [top]
Holmium Oxide is an internationally recognized wavelength calibration primary standard, supported by NIST and other standards organizations. It provides 14 official absorbance bands ranging from 241 nm to 641 nm. Caffeine is considered a secondary standard, with two absorbance bands at 205 nm and 273 nm. Caffeine is also widely used as a wavelength and absorbance standard.

The HSQ Kit™ has been engineered so that the combination of Caffeine with Holmium Oxide, will provide NIST-Traceable wavelength calibration down to 205 nm. First using Holmium Oxide, the detector is calibrated as described in the instructions, at 241 nm and above. Next, the wavelengths of Caffeine are determined at 205 nm and 273 nm. NIST traceability is achieved by the 273 nm band, which overlaps the official NIST range. Assuming that the detector meets at this wavelength, the 205 nm band becomes NIST traceable, and can be used with confidence to ascertain the wavelength accuracy of the detector at this lower wavelength. Using these two standards in combination with each other, provides a means to qualify your detector from 205 nm to 641 nm with full confidence.

PDF Download: NIST-Traceability of Caffeine for Wavelength Qualification

11. Can I get MSDS (Material Safety Data Sheets) for these solutions? [top]
Yes, they are available to all customers that purchase the HSQ Kit™ or you can download them from our website at http://www.mtc-usa.com/msds.asp

12. Can this kit be used to measure the LOQ and LOD differences between instruments in my lab? [top]
Yes, this is another really great “extra” of the HSQ Kit™. By determining the extra column volumes and the dwell volume of each instrument using the HSQ Kit and the software provided, you can determine the differences between instruments.

13. How many injections should I expect on the HSQ Kit HPLC Column? [top]
Thousands... if you do not drop it, overheat it, put too much pressure through it (over 4,000 psi) or inject anything on the column that is not part of the PQ Kit™.

14. What is the acceptable wavelength band width for HPLC detectors? [top]
3nm is the normal setting for wavelength bandwidth for HPLC Detectors. 5nm may be considered too wide and 1nm is too narrow.

15. I currently use a glass filter, why should I use these solutions instead? [top]
Glass filters for uv detectors are not considered primary standards whereas the solutions in the MicroSolv HSQ Kit™ are primary standards.

16. Will the FDA accept the Excel Software in this kit as valid? [top]
The FDA does accept Excel as long as you implement your normal internal controls over it.

17. How do I store the PQ Validation HPLC Column in the HSQ kit? [top]
One of the best features of the MicroSolv HSQ Kit™ is that the kit was designed for ease and speed. The HPLC Column is shipped and stored in the same mobile phase that is used during the testing. The column is always ready to go with minimal equilibration time. The mobile phase/storage solvents were chosen for long column life.

18. How many tests will I get out of one kit? [top]
Depending on how careful you are with transferring the solutions, each kit will produce up to 30 tests. These tests can be on one instrument or on 30 different instruements.

19. What is the mobile phase used in the HSQ Kit and how much is used? [top]
For isocratic system testing, prepare one liter of 13:87 Acetonitrile:0.1% Acetic Acid (1mL/L). For gradient testing, prepare 2 liters.

20. Do the different tests in the kit use the same method conditions? [top]
Yes, this is why it can be run overnight from an autosampler. Simply set your conditions, program the system for PQ Validation and let it run. Use the following Conditions:

Wavelength:	273nm
Injection Volume:	10µl (except for volume linearity)
Flow Rate:	1 mL/minute
Temperature:	Ambient/font>

21. Are column oven temperatures qualified by the HSQ Kit? [top]
No, the use of column ovens are not required for PQ Testing.

22. What is the operating range for temperature with the HSQ Kit? [top]
15°C – 27°C.

23. Having difficulties with wavelength accuracy readings. We are evaluating a number of HPLC's using the HSQ kit. The equipment that we are evaluating is old and has not been undergoing routine PQ. While we are getting passing results for linearity, we are getting confusing results for the wavelength accuracy. In both cases, a new lamp was introduced before the evaluation.

The first instrument did not show any significant absorbance maximum for the primary Caffeine band at 205 nm. From 185 nm to about 240 nm the absorbance reading did not change. We are observing the absorbance readings and were obtaining values of 0.157 from 190 nm to 242 nm. The absorbance then started to change and we did observe a maximum for the second band at 273 nm but the absorbance readings are quite small.

Another similar instrument (same model) showed the primary caffeine band but it was observed at 186 nm rather than at 205 nm. Then when using the Holmium oxide solutions we observed a band at maximum near the expected values for the 537 nm and 451 nm bands but did not observe maximum (+/- 5 nm ) for the 361nm, 267 nm or 241 nm. In fact the absorbance readings for the last three bands were negative — even though we zeroed the equipment 590 nm.

We recently had both of these pieces of equipment cleaned and checked by a service person. He maintains that the confusing results are more likely to be caused by getting air bubbles into the cell following the procedure described than there actually be something wrong with the wavelength. We repeated both results and while the absolute numbers change, the observations are the same.

Could you provide any comments/insights into the data that we are observing? [top]

Evaluation of wavelength accuracy on older, less sensitive detectors can be a little difficult at times. As you mentioned, getting bubbles into the flow cell is a problem. We recommend that you pull the WC solution through the flow cell, then disconnect the syringe, and allow the system to rest, and keep the inlet/outlet tubing ends above the flow cell level, to prevent syphoning. Once everything is stabilized, you can begin the process by auto-zeroing in a region of no absorbance (ca. 590 nm is recommended), then begin the readings.

These older detectors are not the most sensitive, and have wide bandwidths. This will make accurate detection of the lower absorbing, narrow peaks (i.e., 241, 278 and 287 nm bands) difficult. Absorbance readings on the order of 0.1-0.2 AU are typical for these types of detectors, and the differences between the readings near the maxima are not that strong. Your data is not unusual for detectors of this design. This is because the detector is averaging the signals over a broad range of wavelengths. My guess is these instruments have a practical bandwidth of 5-8 nm. You might be forced to look only for the strong, isolated bands such as 641, 537, 451 and 361 nm. The 241 nm band is difficult, as it is sharp, and near a baseline rise at lower wavelengths. An older detector simply may not be capable of resolving the 278 and 287 nm bands, and I use that test as a kind of indicator of the detector resolving power, or lack of.

The 204 band of caffeine should be resolvable, even on an older instrument, while the 273 nm band should be clearly discernable on any instrument. Perhaps the above suggestions of obtaining a more stable signal will help. Also, you are looking for the maximum in a known wavelength region — don't stray too far away from the expected maximum, or you might get enough signal change to falsely indicate another change in the signal direction.

These limitation lie in the instrument, rather than the solutions. I would suggest that you use the results from the strong, available bands, as you have done, and issue the qualification based on those values. In this case, you might be limited to reporting values for the 273, 451 and 537 nm bands, and noting that the instrument was incapable of resolving the finer band structure of Holmium Oxide.

Using the few wavelengths that can be resolved will still produce NIST traceability to the holmium oxide solution.

24. In my older HSQ Kit, the tailing factor minimum was between 0.9 and 1.1, in the latest version, there is no minimum requirement. How does this effect my SOP written with the older kit and is the newer version just as valid and accurate? [top]
The tailing factor reflects the dispersion arising both from the column itself, and from the extra-column (instrument) dispersion. The instrumental dispersion is being measured during the system suitability run, and is part of the PQ calculations. We have taken out this value minimum because it has no effect on the quantitation of the performance of your instrument. The total instrument extra column volume is subtracted from the column efficiency for a true value of the column. The condition of the column itself is reported as the “true column efficiency” under the Extra-Column Volume test protocol. This allows one to distinguish between a failing column, and tailing due to instrumental dispersion. The true column efficiency should be greater than about 8,000 plates if the column is in good condition.

Any SOP’s written can be modified to state that the PQ kit no longer requires a minimum tailing factor as part of system suitability.

25. What is the difference between Verification, Validation and Qualification? [top]
In an LCGC Magazine article by Drs. Ira Krull and Michael Swartz they reference the International Organization for Standardization (ISO) as follows: “Validation is confirmation, through the provision of objective evidence that the requirements for a specific intended use or application have been fulfilled” whereas Verification is confirmation, through the provision of objective evidence that specified requirements have been fulfilled.” They go on to say that if you use a compendial method, you verify and if you a non compendial or original method, you validate.

The term Qualification is now reserved for “instruments” whereas validation and verification are for methods and software.

For more details and further explanations see LCGC North America, Volume 23 Number 10 October 2005, Page 1100.

26. I am getting a different retention time for the peaks in the Resolution Test Mixture on different systems. What impact does this have on the integrity of my data? [top]
No impact at all. There will certainly be mobile phase preparation differences, aging of columns, etc. The HSQ kit instructions call for adjustment of the mobile phase to meet system suitability, which for example in our C of A, calls to adjust the mobile phase to achieve a 4-5 minute retention time window. Keep in mind that the C o A merely confirms that the column was tested, and met specs when it left our facility.

27. What is the effect of extra column volume for different HPLC instruments on the same method? [top]
The difference can be very significant. Extra column volume that is “hidden” in your HPLC system, the fittings, the tubing and the flow cell can greatly impact the efficiency of your method. Click here for an example of the impact.

To determine your extra column volume, use the MicroSolv HSQ Kit for HPLC Self Compliance. Easy to use and low cost.

28. What are the peaks/analytes in the Resolution Test Mixture? [top]
The peaks/analytes elute in the following order for the PQ Kit™:

1. Uracil
2. Theophylinne
3. Caffeine
4. 8-Chloro-Theophylline

1. Uracil
2. Theobromine
3. Theophylinne
4. Caffeine
5. 8-Chloro-Theophylline

29. What is the difference between the new HSQ Kit™ and older PQ Kit™? [top]
The difference between the HSQ Kit™ and PQ Kit™ is mostly speed. HSQ is an acronym for High Speed Qualification. The HSQ Kit™ is supplied with a different, shorter PQ HPLC Column. It is still certified but it is shorter and the run times are much shorter. The time to do the exact same qualification now only takes Ľ the time the PQ Kit™ did. The PQ Kit™ was very fast but our scientists have thoroughly tested the kit with new column and have completely validated all procedures.

Also, the HSQ Kit™ has a different Resolution Test Mixture to better determine the system extra column volume for highly efficient instruments. This extra peak is not required but may be preferred.

Another difference is the "Operating Instructions" or the "User Manual" is greatly consolidated for ease of use. We call it the Quick Start Instructions. In all HSQ Kits, the CD is supplied with both the new Quick Start Instructions and the previous PQ Kit Instruction Manual which has a lot of reference information. Of course, the unlimited technical support is the same for both kits.

30. I have my SOP written with the older PQ Kit™ using the longer column. Will you still sell it and support it? [top]
Yes, absolutely! The PQ Kits and the longer columns are still available. The replacement solutions are the exact same for both kits.

31. Can the L3 linearity solution be used for wavelength accuracy? [top]
The short answer is yes. Many other vendors use only caffeine for wavelength accuracy, at 205 and 273 nm. Holmium oxide of course covers 241-641nm, and is directly traceable to NIST standards. However, if you want, there is no reason you could not simply take the spectrum of caffeine.

The L3 solution provides a nice, mid-range absorbance value, compatible with most UV-Vis detectors. You could of course use any other concentration that gives a suitable response. The reference solution would be mobile phase, but there is very little difference if wanted wanted to use water.

32. The instructions which are given in the PDF file and on the Excel sheets are very detailed and a bit overwhelming from my perspective. What should I do? How can you help? [top]
I can sympathize with you somewhat, as the first time through, it can be a bit overwhelming. However, if you simply examine the data entry section of the Excel sheet, the items that need to be entered are in order of their injection.

There are 3 basic methods to be written, and 3 sequential sections of the injection run sequence.

1. System Suitability
   Method RTM. Inject one or more blanks, and the RTM. Check that the retention time and efficiencies meet specs.
   
   
2. Isocratic
   Method PQ. Inject blanks, the 10 precision samples of L3, the 6 linearity standards L1-L6 each in triplicate, then three blanks (to measure carryover). Continue with the injection volume linearity (assuming she has a suitable autosampler) injecting triplicates of L2 at each of 5 chosen injection volumes.
   
   Done. This is all there is to the isocratic testing.
   
   
3. Gradient
   If you have a gradient system to test, then set up method GRD, with the exact profiles as detailed in the Quick Start instructions. Spike about 500 mL of mp with the GVS solution (3 mL GVS per 500 mL mp), and flush the LC system, putting the spiked mp in the "B" position.
   
   Inject a sample of the actual spiked B mobile phase at the start. Run the gradient profile.
   
   At the conclusion, there will be a small void volume peak (Uracil) from the spiked B injection. That is the column void volume. Using an expanded scale, estimate the onset of the gradient. Those two values are entered into the spreadsheet, to calculate the Gradient Dwell Volume.
   
   Use a ruler, or integration to measure the heights of the 10% and 90% gradient steps for A/B, C/B and D/B. Enter those values in the spreadsheet, for calculation of the gradient accuracy. Note that I recommend 2 injections of the gradient, using only the second one for the qualification data, since it is difficult to fully purge the system on the first try.