January 2011 saw the publication of the new revision of European Union (EU) GMP Annex 11 on Computerized Systems and Chapter 4 on Documentation. What will the impact be of these two revised regulations on computerized chromatography systems and  chromatography data systems (CDSs) used in regulated GMP laboratories?

Regulations and guidance for the pharmaceutical industry have been changing at an increasing rate over the past 10 years. The latest of these was from the European Medicines Agency (EMA) in January 2011 and was the long awaited revision of Annex 11 on computerized systems together with consequential changes in Chapter 4 on documentation.

  These two updates become effective on 30 June 2011. This column looks at the major changes in the two revisions and explores their impact on any regulated GMP laboratory that has to comply with these regulations. This will be a selective look and readers are encouraged to look at the EMA web site to read the whole of these documents.

  The question posed by the title is "Is Annex 11 Europe's answer to 21 CFR 11 on electronic records and electronic signatures?"

  We will explore this question as we analyse the new regulations. 

  is structured very differently from United States GMP for pharmaceuticals: European GMP is split into three parts plus 20 annexes. Part 1 is concerned with finished pharmaceutical products and Part 2 with active pharmaceutical ingredients (this is ICH Q7

  adapted into European law). The annexes are regulations on specific topics applicable to both Parts 1 and 2 with Annex 11 focused on computerized systems.  

Annex 11 has been part of EU regulations since 1992 and unchanged until now. Since it was published there have been many technology changes but also organizational changes such as outsourcing and software as a service (SaaS). In 2008, a proposed draft of Annex 11 was published for industry comment and over 1400 replies were received by the EMA. In January's version, many of the more wacky proposals in the 2008 draft have been removed or toned down. However, the new version is an expansion of the current regulation and there are new requirements that laboratories must consider when working under these regulations or exporting medicines to Europe.  

I will give an overview of the changes that will be effective from the end of June 2011 in this article. Owing to the amount of space available this will not be a comprehensive discussion of all changes so please read the whole document yourself.

  The various sections and topics of the new version of Annex 11 are presented in Table 1. 

 Table 1: The structure and content of the revised EU GMP Annex 11. 

The first section to discuss is the Principle that covers the scope and application of the new regulation. The key elements of the Principle are:  

Annex 11 applies to all forms of computerized systems used as part of GMP-regulated activities.

This is a wide scope statement and includes all laboratory computerized systems such as chromatography data systems (CDSs), plus the spreadsheets and databases developed in your laboratories, as well as statistical analysis software programs.  

The application should be validated; IT infrastructure should be qualified.

This is the major item: the ten words that mean so much — validate applications and qualify infrastructure. Validate applications — OK, this has been a given for the last 25 years, although we tend to consider systems which includes the infrastructure. However, for the first time in any regulation there is a specific and explicit requirement for IT infrastructure qualification. Many regulated organizations do qualify infrastructure, but there is now a regulatory requirement to do so. For the companies that have not done so this becomes a major retrospective qualification exercise.   

When a computerized system replaces a manual process there should be no resultant decrease in product quality, process control or quality assurance.

In a carry over from the current version of Annex 11 with the additional requirement of process control systems that may be relevant when a chromatograph and CDS is used in a Process Analytical Technology (PAT) project. In essence, it requires that as a minimum, the quality of a computerized system should be as good as the manual system it replaces. In reality, the computerized system should be much better because the human factor (i.e., the liveware that makes most of the mistakes), is usually taken out of the process. 

Risk Management throughout the Life Cycle

Annex 11 has always had risk management but it was buried in the section on validation of systems: "the amount of validation work required was dependent on the nature of the software and if novel elements (i.e., custom software) were incorporated". Now we have a new section requiring risk management to be applied throughout the life cycle of a computerized system to ensure patient safety, data integrity and product quality. The work done should be based on a "justified and documented risk assessment". Hmm, I wonder where this wording came from? That's right, the Europeans have stolen the phrase from the FDA's Part 11 scope and application guidance!

  The good side of this is that we are getting harmonization of regulations which can only be a good thing. There is a further and specific mention of risk management under the new section on change control and configuration management. 

In section 2 on personnel there still remains the requirement for close cooperation between all involved with the system including IT from the original version of the regulation. However. we have new two new roles mentioned in the text and defined in the glossary:   

: the person responsible for the business process. The individual in this case would typically be a senior manager as a business process may impact more than one department.

: the person responsible for the availability and maintenance of a computerized system and the security of the data residing on that system (typically a senior spectroscopist or laboratory manager). Typically, the person who goes to jail if the system validation is wrong or had not been done.

This area is a major expansion of Annex 11 that moves from a single sentence into four clauses in an attempt to catch up with technology and organisational changes that have occurred since the original version was issued. When third parties are used to carry out any work (supply of product or service) on a computerized system there needs to be a formal agreement (contract or service level agreement). Clause 3.1 notes that there should be "clear statements of the responsibilities of the third party". Then there is a short sentence stating "IT departments should be considered analogous" which means that even if an organization's own IT department is used to support a validated computerized system, there needs to be a contract or service level agreement (SLA) in place with the regulated laboratory. There is also the requirement for audits of a supplier or service provider, with the decision for this being based on a risk assessment, which, of course, will be documented and approved. The issue of cloud computing, if used by a regulated laboratory, may be partially addressed though service contracts but how will the requirement for IT infrastructure to be qualified be met? An interesting issue that may prevent take up by conservative pharmaceutical companies.  

When an audit of a supplier or service provider is performed, the new regulation requires that the "quality system and audit information" is available to inspectors on request. This is a major departure from current practices, typically an audit report is seen as an internal quality assurance (QA) document by many companies and only the evidence that an audit took place will be a certificate given to an inspector. However, currently there is major regulatory concern with the quality of the whole pharmaceutical supply chain, which includes software and services. The European regulators are taking a hard line and want to read the supplier and service provider audit reports to satisfy themselves that the service of product for critical operations has the quality built into it.  

Gazing at my crystal ball, I think that an implicit requirement in this new regulation will be the emergence of vendor management

  where pharmaceutical companies will monitor suppliers to ensure that corrective actions following audits of quality management systems, products and services have been implemented effectively. For supplier and service providers where lip service is paid to quality it will also mean increased number of audits by the same customer to ensure corrective actions have been completed (e.g., follow-up audits over perhaps a number of years). For further information about managing the risks associated with the software supply chain, I would suggest reading a report, published in December 2010, from the Software Engineering Institute (SEI) at Carnegie Mellon University.

  Some of the main conclusions of this report are:  

Product development is completed in advance of an acquirer's product and supplier assessment.

There is no guarantee that current supplier development practices were used for a specific product. 

For custom system acquisitions, acquirers can and should actively monitor both contractor and product supply chain risks during development. 

This report suggests contractor and acquirer activities that support the management of software supply chain risks.

Finally in this section, any documentation supplied for commercial off the shelf products needs to be reviewed to see that user requirements are fulfilled (this is not the user manuals by the way). Hmm, I suggest you read an earlier "Focus on Quality" column from

magazine, about my views on material supplied by vendors in this area

  as it can often be quicker to write your own user requirements. 

The validation section has been expanded from one to eight clauses in the new version. The key changes are that a life cycle should be used to validate a system and that manufacturers should be able to justify their approaches based on risk assessment. The Annex 11 update does not mandate any validation approach but whichever one is selected for a specific system it needs to be justified and documented to withstand regulatory scrutiny. Some administrative requirements for validation are an inventory for computerized systems, although this would have been useful to link with the validation master plan

  For critical systems there needs to be a current system description. In effect, the new requirement for an inventory formalizes what is usually required for an inspection and the system description is now limited to critical rather than all systems, as required by the old version of the regulation.  

For each computerized system validation there needs to be a user requirements specification to describe the required functions of the system based on risk assessment and GMP impact.  

Furthermore, there is now the need for requirements traceability throughout the life cycle, again for the first time in a regulation a traceability matrix

 is required. The test methods and test scenarios need to be documented and testing should include the overall process with consideration of data limits, parameter limits and error handling. The latter is particularly important to know before a system becomes operational, rather than when discussing this with an inspector. Annex 11 also allows the use of automated test tools and test environments, providing that they have documented assessments for their adequacy for the task. Before you all rush off and spend money on automated test tools bear in mind an assessment by Frewster and Graham

  that you need to be able to operate a test tool between 8 and 11 times before you break even on your investment. There will be very few laboratory systems that will require automated testing. 

Sections 5, 6, 8, 9 and 12 cover the main elements of data integrity (data, accuracy, audit trails and security) in the new Annex 11. In summary, these sections are looking for checks for correct and secure entry (both manually entered and automatically captured data) and the subsequent data processing to minimize the risks of a wrong decision based on wrong results.  

The identities and access privileges of authorized individuals carrying out work need to be recorded and maintained for each validated system. Further controls are required to secure data by both physical and electronic means against damage and stored data needs to be checked for accessibility, readability and accuracy and this applies to both paper and electronic records. 

Audit trails are not mandatory for all computerized systems but their implementation should be based on a documented risk assessment. Personally, I think that if you are working electronically, then an audit trail is essential for ensuring data integrity. Mirroring some of the recent FDA warning letters, the new Annex 11 requires audit trails to be "available and convertible to a generally intelligible form and be regularly reviewed". The problem is that many audit trails implemented for commercial laboratory systems are simply depositories of unintelligible rubbish; moreover, how will a vendor implement a function in their system to meet the requirement that an audit trail has been reviewed? In addition, the audit trail needs to include the date and time stamps of record entries, changes and deletions which brings the EU regulation close to the US Part 11 requirements on the same subject.  

Print outs, both of electronically stored data and any records used to support batch release, need to be available. There is also a further and specific requirement for any print out supporting batch release to indicate if any data has been changed since the original entry. This is so that the Qualified Person (under EU GMP a batch can only be released by suitably trained individual called a Qualified Person or QP) can check what changes have occurred. However, most vendors will point to the audit trail search function as the means to fulfil this requirement.

 This is inadequate. What is required is that when the result is printed out, there is an annotation or equivalent to indicate if the result has been changed or not. Chromatography Data Systems (CDSs) have this for baseline fits: unchanged baseline fits are in capital letters and manually changed ones are in lower case. 

In addition, there are requirements for data migration (section 4.8) and archiving (section 17) to ensure that electronic records acquired in one version of software can be read in a new version as well as allowing data to be archived. However in the latter case, the data should be assessed for "accessibility, readability and integrity" especially after changes made to the backup software or the system itself. 

The new version of Annex 11 also sees the formalization of electronic signatures in EU GMP. Many laboratories have implemented electronic signatures based on 21 CFR 11

 but Annex 11 does not appear as stringent or as overly bureaucratic as the US regulation. The European requirements for electronic signatures simply state that electronic signatures are to have the same impact as hand written signatures within the boundaries of the company, be permanently linked to the respective record and include the time and date that a signature was applied. There is not the heavy bureaucracy and formality of 21 CFR 11 to send letters to the FDA, nor is there the need to have training in nonrepudiation of an electronic signature or description of the three different types of signature. However many of the same requirements are implicit, as the European legislation simply states that electronic signatures have the same impact as handwritten signatures and hence all of the nonrepudiation requirements apply immediately. The advantage of the European legislation is that practicing inspectors have drafted the regulation rather than lawyers. Perhaps if the FDA ever gets around to reissuing Part 11, could it look and read like Annex 11? Now that IS an interesting thought. 

The current Annex 11 IT requirements of backup, security, incident management and business continuity have been carried over to the new version and expanded. Backups (section 7.2) need to be performed regularly but the new version has expanded requirements for checks for the integrity and accuracy of backup data (which, of course, will be documented) and the ability to restore data that is checked during a system validation and also periodically thereafter (you guessed it — which is also documented). This is intended to ensure that backup media can still be read throughout the record retention period. The security section also includes the network as well as individual applications so the extent of controls depends on the criticality of the application but also if you are accessing it from inside or outside an organization. 

Incident management has changed from a simple statement of "any failures and remedial action should be recorded" to "all incidents, not only system failures and data errors, should be reported and assessed". So the scope has been widened greatly. However, the new version goes further "The root cause of a critical incident should be identified and should form the basis of corrective and preventative actions". So implied within this process should be a means to assess and classify errors and then for the critical ones only to undertake a root cause analysis and then formulate both corrective and preventative action plans. It is this portion of the new regulation that will impact many incident management processes and have the IT department scrabbling to understand root cause analysis. 

Again, business continuity was covered in clauses 15 and 16 in the old regulation which have been consolidated into clause 16 in the new version. What is required is to have plans available to ensure continuity of support for critical processes as well as knowing the time required to bring alternatives into operation based on risk assessments. However the new regulation specifically requires that these arrangements be documented and tested adequately before use. There is no use having a business continuity plan that fails as the last set of backup tapes are corrupted and that your alternative computer site is not available when you need it, or perhaps the plan was written and has not been updated to account for the latest technology.  

Articles

Questions of Quality

How will the new revision of EU GMP Annex 11 on Computerized Systems and Chapter 4 on Documentation affect chromatographers?

Is GMP Annex 11 Europe's Answer to 21 CFR 11?

Microextraction by packed sorbent (MEPS) is a new technique for sample preparation that can be connected on-line with liquid chromatography (LC) or gas chromatography (GC) systems without any modifications. This article describes the use of MEPS in clinical and pre-clinical studies to quantify different drugs in whole blood samples. MEPS was used to determine cyclophosphamide in mouse blood from pre-clinical studies using 20 µL of blood samples. The inter-day accuracies and precisions ranged from 107–109% and from 2.0–7.0%, respectively. The determination of four immunosuppressive drugs in human blood by MEPS and liquid chromatography–mass spectrometry (LC–MS) is described. The method showed a good selectivity and sensitivity. The calibration curves for everolimus, sirolimus and tacrolimus ranged from 0.5–50 ng/mL and for cyclosporine was 3.0–1500 ng/mL. Intra-day precisions for the studied immunosuppressive drugs were 2.0–11.7% and inter-day precision ranged from 5.1–13.7% (CV).

Off-line sample preparation is a limiting step for achieving high-throughput bioanalysis, indicating a clear need for on-line sample preparation to speed-up the process. Solid-phase extraction (SPE) is attractive for recovering drug residues because of its ability to efficiently retain highly functionalized compounds from aqueous samples and then release them into organic solvents on elution. SPE methods are useful for complex biological samples because the main requirements of the extraction (matrix exchange, desalting, removal of macromolecules and highly polar compounds) are well matched to the properties of the sorbent. When conventional SPE was introduced to the market, it was well accepted by those studying metabolism and toxicology because of the superior efficiency and selectivity that the technique offered over liquid-liquid partitioning methods. Liquid-liquid extraction (LLE) methods require solvent or solvent mixtures of fairly high polarity and these often yield emulsions and high matrix background in the extracted sample. 

For even more polar analytes, such as drug conjugates and metabolites, recovery and sample purity is further compromised. The same reasons that made conventional SPE so attractive to the study of drug and toxin residues are equally applicable to microextraction by packed sorbent (MEPS) because both are based on the same sorbent chemistries. MEPS uses the same sorbents as conventional SPE columns and so is suitable for use with most existing methods by scaling down the reagent and sample volumes. The excellent performance of MEPS was recently illustrated by on-line LC–MS and GC–MS assays of drugs and metabolites in water, urine, plasma and whole blood samples.

In this article, the application of MEPS for the determination of drugs in whole blood samples will be described and the unique characteristics that make MEPS a credible alternative to conventional SPE in this area will be discussed.  

The Role of Sample Preparation in Bioanalysis

The matrix problems may call for specific skills in quantitative bioanalysis, particularly in electrospray ionization-mass spectrometric detection (ESI-MS). Signal suppression caused by unknown matrix interferences is often observed and can give rise to incorrect data interpretation, such as poor accuracy and precision. Therefore, the sample preparation is an important step in bioanalysis processing to avoid ion suppression phenomenon. The sample preparation methods as the first step in an analytical procedure are very important for total analysis processing. If an unsuitable sample preparation method has been used before the injection, the whole analytical process can be wasted. The purpose of the sample preparation is 1) the removal of specific interferences; 2) the conversion of the analytes into a more suitable form for injection, separation and detection; 3) the pre-concentration (enrichment) of the analytes to improve the method sensitivity; and 4) to eliminate ion suppression in ESI-MS.  

An ideal sample preparation method should a) involve a minimum number of working steps; b) be fully automated; and c) the procedure must be highly reproducible with a high recovery of the target analytes. Because of the low concentration levels of drug in plasma and the variety of the metabolites, the selected extraction technique should be virtually exhaustive. Commonly used sample preparation methods in pharmaceutical industry are SPE, LLE and protein precipitation. The growing number of samples to be analysed requires high-throughput and fully automated analytical techniques.  

Recent developments of sample handling techniques are directed, from one side, toward automatization and on-line coupling of sample preparation units and detection systems and from another toward development of more selective sorbents, such as molecular imprinted polymers (MIPs) and restricted access materials (RAMs). LLE and SPE are the most important sample preparation methods in bioanalysis. SPE methods are effective for complex biological samples because the main requirements of the extraction (matrix exchange, desalting, removal of macromolecules and highly polar compounds) are well matched to the properties of the sorbent. MEPS is a logical extension of SPE for handling biofluids because the small operating volumes reduce the size of sampling. Reducing the demand for sample can extend the use of MEPS to experiments that were previously beyond the scope of SPE (e.g., multiple collections of fluid from small animals or volume limited 

 experiments, confirmatory testing of multiple drug residues in hair, saliva or CSF) as well as making the sample collection less invasive. 

Microextraction by packed sorbent (MEPS) is a new embodiment of SPE that has been miniaturized to work with sample volumes as small as 10 µL and larger (up to 250 µL). MEPS uses the same sorbents as conventional SPE columns, but unlike conventional SPE columns, the MEPS sorbent bed is integrated into a liquid handling syringe that allows for low void volume sample manipulations — either manually or in combination with laboratory robotics. MEPS allows sample processing, extraction and injection steps to be fully automated as an on-line/at-line sampling/injecting device to GC or LC. 

In MEPS the sorbent, 1–4 mg, is either inserted into the syringe (100–250 µL) barrel as a plug or between the needle and the barrel as a cartridge. The cartridge bed can be packed or coated to provide selective and suitable sampling conditions. Sorbent materials such as silica-based materials (C2, C8, C18), strong cation exchangers (SCXs) using sulphonic acid-bonded silica, RAMs, HILIC carbon, polystyrene-divinylbenzene copolymers (PS-DVBs) or MIPs can be used. MEPS was invented and developed at AstraZeneca, Södertälje, Sweden.

While the capacity for truly miniaturized extractions allows sparing use of sample and reagents, a more significant strength of MEPS is the ability to fully elute the sorbent with volumes of 20–50 µL through a syringe needle. Such capability allows MEPS to be adapted for on-line use with both GC and LC inlets or in 96-well plate format for use with immunoassay and other colorimetric techniques. 

MEPS extraction steps are the same as for standard SPE: extraction, washing and elution have to be optimized to reach the highest analyte recovery. In MEPS extraction procedures, additional steps for post-cleaning and re-conditioning have to be included to enable multiple uses of MEPS sorbent. Extraction protocol is presented in Figure 1 for C8 and C18 as sorbents and in Figure 2 for mixed-mode sorbents. Blood samples need to be diluted 20–25 times with water or acidic water whereas plasma samples need to be diluted five times. Using mixed mode (anion–cation exchange) the sample pH has to be adjusted to get charged analytes. The first stage is sample loading and can be done once or more depending on sample concentration and if pre-concentration of the sample is required. In general four sample-loads (4 X 100 µL) is enough for the pre-concentration of analytes and to achieve a good recovery. In addition, the speed of the plunger movement can range from 10–20 µL per second to get a high extraction recovery. 

 Figure 1: MEPS extraction protocol for blood and plasma samples using C4, C8 or C18 sobents. 

The second stage is a washing step and this is done with 100 µL of a 0.1% HCOOH in 5% isopropanol–water solution (or with 5% methanol in water). The third stage is the elution step and in this step pure methanol can be used (for on-line GC injection) or a mixture of methanol/acetonitrile and water at the correct pH [for reversed-phase (RP) LC injection]. Furthermore, for mixed-mode MEPS, the pH plays an important role (control charged/uncharged analyte) in achieving a high recovery. In addition, the best possible elution solvent should elute the analyte using the smallest volume of solvent possible. The elution volume can range from 20–50 µL and can be eluted in one step (50 µL directly) or by two steps (2 X 25 µL). Finally, a post-clean step after the extraction procedures in order to clean the sorbent to avoid carry-over and to re-use the MEPS sorbent can be performed by weak and strong washing. The weak washing can consist of water (90%) and small amount of solvent such as methanol or

-propanol (10%). The strong washing can be 0.2% ammonium hydroxide in methanol or acetonitrile — or a mixture of both. 

Figure 2: MEPS extraction protocol for blood and plasma samples using mixed-mode sorbent (anionâcation exchange) for acids (pK = 2â8) and bases (pK = 2â10). 

A key step for any analytical protocol is the determination of the optimum analyte recovery for the overall method and for each individual procedure. The extraction protocols for C4, C8 and C18 may need adjustments for some analytes that are highly lipophilic or hydrophilic by altering the polarity or pH of used solvents. In these cases the wash and elution steps may need some modifications and tetrahydrofuran or hexane can be used as the elution solution for highly lipophilic compounds.  

Comparison of MEPS with Other Extraction Techniques

MEPS differs from SPE because the sorbent bed in MEPS is integrated into a syringe that allows low void volume sample manipulations — either manually or automatically — in combination with an autosampler. Additionally, MEPS uses sparse amounts of sample and reagents, but a more significant strength is the ability to fully elute the sorbent with volumes of less than 10 µL. MEPS is simple, rapid and does not consume large amounts of solvent compared with SPE and LLE methods, and can achieve the same sensitivity. 

While solid phase microextraction (SPME) is suitable for on-line use in combination with GC or LC methods, its ability to handle low concentrated analytes in small sample volumes (1–2 mL) is limited. In SPME, the sampling fibre is quite sensitive for the nature of sample matrix such as urine, blood or plasma. MEPS can be used for these complex matrices. Sébastien Anizan et al. evaluated SPME and MEPS as extraction techniques for the analysis of steroid metabolites in urine by GC–MS.

  Anizan found that in this case SPME fibre had a short lifetime. After only five extractions of urine samples, a degradation of the SPME fibre was observed. In addition they reported that MEPS was performing rapidly, repeatably, and with a high throughput. They reported that the SPME extraction time was 60 min and the recovery was below 11% while MEPS recovery was 60–98% with an extraction time of 3 min for all model compounds. 

The differences between MEPS and Zip-tips (packed tips) is that MEPS can be adapted for on-line use with both GC and LC inlets or in 96-well plate format for use with immunoassay and other colorimetric techniques. Additionally, the MEPS sorbent bed can be used for at least 100 extractions — not only once.  

Stir bar sportive extraction (SBSE) is regarded as being more difficult to automate, but semi-automated protocols have been described in the literature. SBSE has a good recovery but has a long extraction time of 30 min or more compared with 1–3 min for MEPS.  

Determination of cyclophosphamide in mouse blood:

Cyclophosphamide is one of the most used anti-cancer drugs and is used in the treatment of malignant diseases such as leukaemia, lymphoma and solid tumours. In this study MEPS was used to extract cyclophosphamide from mouse blood in a preclinical study.

20 µL of blood samples, standards and QC samples, were diluted with water (1:25). The MEPS sorbent (C2, 1.0 mg) was first conditioned with 50 µL methanol followed by 50 µL of water. The spiked blood and blood samples were withdrawn into the syringe by the autosampler (4 X 100 µL). The sorbent was then washed once with 100 µL of water/methanol 95:5, (v/v) to remove proteins and other interferences. The analytes were then desorbed by 40 µL methanol/water 90:10 (v/v) directly into the LC injector. Cleaning of the sorbent was carried out using 4 X 250 µL elution solution followed by 4 X 250 µL of the washing solution between every extraction. The same packing bed was used for about 100–120 extractions before it was discarded.  

A gradient HPLC was used. Mobile phase A was 0.1% formic acid in water and acetonitrile 90:10 (v/v) and mobile phase B contained 0.1% formic acid in water and acetonitrile 20:80 (v/v). The gradient started from 1.0% of phase B up to 50% from 1–4 min and then from 4–6 min to 90% of phase B and at 6.5 min phase B was set at 1.0% again. All experiments were conducted using a triple quadrupole mass spectrometric instrument: the Micromass Ultima (Waters Corporation, Manchester, UK). The MEPS syringe and cartridge was obtained from SGE Analytical Science (Melbourne, Australia).  

Calibration curves of mouse blood samples spiked with cyclophosphamide standards were obtained in the range 0.1–100 µg/mL. The coefficients of determination (r) were > 0.99 for all runs. Figure 3 shows standard curve of the cyclophosphamide in mouse blood using MEPS–LC–MS–MS.

Figure 3: A typical standard curve of cyclophosphamide in whole mouse blood.

The inter-day mean accuracy ranged from 107–109% using MEPS–LC–MS–MS. The data of inter-day variation of the precision (CV) were in the range 2.0–7.0%. The data of withinbatch variation of the precision (RSD) were in the range 4.0–6.0%. The accuracy and precision results are summarized in Table 1. The carry-over was less than 0.4% after the first blank and after the third blank it was negligible. The method was used for the determination of cyclophosphamide in mouse blood from pre-clinical studies.

Table 1: Intra- and inter-day precision for cyclophosphamide in mouse blood using MEPSâLCâMSâMS.

Determination of Immunosuppressive Drugs in Human Blood:

 Four immunosuppressive drugs, cyclosporine, everolimus, sirolimus and tacrolimus in human blood were extracted and determined by MEPS followed by LC–MS–MS.

 In this study, different kinds of sorbent material were investigated and the highest recoveries were obtained with C8 sorbent compared with C18 and C4. Human blood (50 µL) spiked with cyclosporine, everolimus, sirolimus and tacrolimus was first diluted 25 times with 0.1% formic acid in water and then extracted on a C18 MEPS-cartridge (4 X 100 µL sample loading). The sorbent was then washed once with 100 µL of 5% methanol in water. The analytes were then desorbed by 50 µL methanol/isopropanol/ acetonitrile/water (50:30:10:10). Cleaning of the sorbent was carried out using 4 X 250 µL elution solution (methanol/isopropanol/ acetonitrile/water) followed by four 250 µL of the washing solution (5% methanol in 0.1% formic acid) after every extraction. This step decreased memory effects, but also functioned as conditioning step before the next extraction. The same packing bed was used for 100 extractions before it was discarded. 

A gradient LC coupled to a triple quadrupole mass spectrometer was used and the total run time for each sample was 2.5 min. The method showed a good selectivity for all analytes. The calibration curves for everolimus, sirolimus and tacrolimus ranged from 0.5–50 ng/mL and for cyclosporine was 3.0–500 ng/mL. Representative mass chromatograms of blank human blood and spiked human blood with cyclosporine (CS) are presented in Figure 4.  

Figure 4: SRM chromatograms obtained from blank human blood and spiked human blood sample with cyclosporine at  500 ng/mL. 

Intra-day precisions for the studied analytes were 2.0–11.7% (CV) and inter-day precision ranged from 5.1–13.7% (CV). The accuracy values were 102–109%. The mean extraction yield was found to be 97% for cyclosporine, 92% for everolimus, 90% for sirolimus and 96% for tacrolimus. The method carry-over was 0.02% or less.  

The MEPS–LC–MS–MS was compared with protein precipitation followed by LC–MS–MS that was used as a routine method. Patient's samples were analysed using both methods. The results showed a good agreement between MEPS and protein precipitation but MEPS gave cleaner extracts and no matrix effects were observed. In addition the sensitivity was improved with MEPS as a result of a pre-concentration effect on the MEPS sorbent. MEPS showed better robustness of the chromatography and MS system compared with protein precipitation method.  

Determination of Local Anaesthetics in Human Blood:

Sample preparation for blood samples is more often concerned with achieving a sufficiently high enrichment of analyte so that detection is possible. In this study

  human blood (50 µL) spiked with lidocaine, ropivacaine and bupivacaine was extracted on a C18 MEPS-cartridge conditioned with methanol (50 µL) and water (50 µL). The sorbent was then washed once with 100 µL of water. The analytes were then desorbed by 50 µL methanol/water 60:40 (v/v) containing 0.25% ammonium hydroxide directly into the injector. Cleaning of the sorbent was carried out using 3 X 250 µL elution solution [methanol/water 60:40 (v/v) containing 0.25% ammonium] followed by 3 X 250 µL of the washing solution (water) between every extraction. This step decreased memory effects, but also functioned as conditioning step before the next extraction. The same packing bed was used for about of 50 extractions before it was discarded. A Zorbax (50 X 2.1 mm, SB-C8, 3.5 mm) column (Agilent Technologies, California, USA) was used as the analytical column and gradient LC was used. Mobile phase A was 0.1% formic acid in acetonitrile and water (10:90) and mobile phase B contained 0.1% formic acid in acetonitrile and water (80:20). The gradient started from 10% of phase B up to 80% from 1–4 min and then from 4–5 min isocratic at 80% of phase B and at 5.1 min phase B was set at 10% again. The LC was connected to a triplequadrupole, mass-spectrometric instrument. 

Table 2: Intra- and inter-day precision for lidocaine, ropivacaine and bupivacaine in human blood at various concentrations using MEPSâLCâMSâMS.  

The calibration curves in human blood were used for the analytes in the range of 10–10000 nmol/L. The method was validated using [

]-lidocaine as the internal standard (IS). Correlation coefficient (r) values obtained were over 0.995. The intra-day precision values are 1.0–3.0% for lidocaine, 2.0–4.0% for ropivacaine and from 2.0–5.0% for bupivacaine. The inter-day precision values are 2.0–7%, 2.0–12% and 5.0–11% for lidocaine, ropivacaine and bupivacaine, respectively. The accuracy varied from 90–109% for lidocaine, from 85–97% for ropivacaine and from 89–97% for bupivacaine (Table 2). The accuracy and precision were well in line with the international criteria. The validation of the methodology showed that the method is highly selective for human blood samples. 

The extraction recovery was about 90% for all analytes (MEPS sampling, 3 X 100 µL). Figure 5 shows a mass chromatogram of direct injection of lidocaine, ropivacaine and bupivacaine in the mobile phase, and blood sample after MEPS extraction. 

Figure 5: Comparison of mass chromatograms of lidocaine (1000 nmol/L), ropivacaine (500 nmol/L), and bupivacaine (500 nmol/L) obtained by direct injection (in the mobile phase) and after extraction from spiked human blood by MEPS. 

In this study the carry-over was investigated by injecting elution solution after the highest standard concentration; the carry-over was about 0.02% after washing five times with methanol containing 0.2% ammonium hydroxide and five times with water. 

Determination of a New pro-drug: Neuronal Nicotinic Receptor Partial Agonist in Human Blood:

 A neuronal nicotinic receptor (α4β2 NNR) agonist for treatment of cognitive deficits in Alzheimer's disease (ADHD) is determined in human blood by MEPS–LC–MS–MS.

A mixed-mode sorbent (polystyrene ENV+) and silicabased ion-exchange (SCX) were used. 20 µL of blood samples were diluted 25 times with 0.1 M ammonium format. The MEPS sorbent was washed first with 100 µL 0.1 M ammonium formate. 100 µL of diluted blood was loaded and the sorbent was then washed once with water (100 µL). The pro-drug was desorbed by 50 µL methanol/water 60:40 (v/v) containing 3% ammonium hydroxides and then 3 µL were injected into the UPLC injector which was coupled to Xevo-QT mass spectrometer (Waters, Milford, Massachusetts, USA). The separation was performed on an Acquity (Waters). column (C18, 1.7 µm, 2.1 X 50 mm) was used. 

Figure 6: MRM mass chromatogram at LLOQ (2 nmol/L) of a new pro-drug (neuronal nicotinic receptor agonist) obtained from spiked human blood. 

Article

Microextraction by packed sorbent is a sample preparation technique that can be connected on-line with LC or GC systems.

On-line Whole Blood Analysis using Microextraction by Packed Syringe (MEPS) and LC–MS–MS

Can pressure alone affect the appearance of a chromatogram?

 articles we've been looking at various factors that can influence selectivity — that is, peak spacing — in a liquid chromatographic (LC) separation. An understanding of these variables can be useful if we are developing a new method or modifying an old one, as well as for troubleshooting existing separations. We started out by looking at the effect of the solvent type on peak spacing.

  We saw that changing from one solvent to another could make dramatic changes in the appearance of the chromatogram. In the next article,

  we found that changing the solvent strength, or percentage of organic solvent, was sufficiently effective to make the necessary selectivity changes to improve the separation.  

Many workers like to use changes in the chemistry of the LC column to change the chromatogram, and this was the subject of the third article in this series.

  makes it possible to improve our chances of picking a column that is very similar to — or quite different from — the column that we are currently using. This month, we'll consider another factor that can affect selectivity — pressure. 

Most of us consider the pressure that we observe in an LC separation to be an artifact of the separation conditions, and not much more than that. In other words, if we double the flow-rate, we expect the back-pressure to double, but that's all. And that is a fairly reasonable conclusion when we consider the pressure changes that have been made historically to LC methods. For example, we might change the flow-rate from 1.0 mL/min to 1.5 mL/min and see that the pressure goes up by 50%. Or we might switch from a 150 mm long column to a 250 mm column, and the pressure will rise by two thirds (250/150). If some other minor changes in the separation occur, they often are ignored. 

What about changes in particle size? Let's say we move from a 150 X 4.6 mm, 5 µm particle column to a 100 X 4.6 mm, 3 µm column in an effort to speed up the separation. The change in pressure is inversely related to the square of the particle size, so the pressure change due to particle size alone should be (5/3)

  ≈ 2.8-fold increase. However, shortening the column should drop the pressure by 100/150 = 67%. The combination should be the product of these two changes, or ≈ 1.9 times increase in pressure. If we see any small changes in peak spacing, we attribute that to minor changes in column chemistry when we change particle sizes — after all, the two packing materials have a little different history in their synthesis, right? 

With the advent of ultra-high pressure liquid chromatography (UHPLC), the changes in pressure that we observe when changing from one condition to another can be much larger than with the particle size changes noted above. For example, we might move a conventional LC method to UHPLC in an effort to reduce the run time. Or, we might use UHPLC as a development tool, but then migrate the method to a conventional system for routine use.  

In such cases, large changes in pressure can be observed. In the example of Figure 1, a method was developed on a 50 X 2.1 mm, 1.7 µm particle column operated at 1 mL/min, which generated ≈ 11000 psi of back-pressure. When the method was converted to a conventional LC system, a 100 X 2.1 mm, 3.5 µm particle column was run at 0.486 mL/min, and the pressure dropped to ≈ 3000 psi. Let's see if this pressure change is reasonable. The 50 mm to 100 mm column length should double pressure. The 1.7 µm to 3.5 µm particle change should result in a drop of (1.7/3.5)

  ≈ 4.3-fold. The flow-rate change should drop the pressure by two-fold. Combined, this should give 11000 psi X 2 X 1/4.3 X 1/2 ≈ 2500 psi, which is close enough to the observed ≈ 3000 psi pressure. The resulting method was suitable for its intended purpose of measuring impurities in a pharmaceutical product, providing adequate resolution of nine potential impurities and the active pharmaceutical ingredient (API). However, a close look at the chromatogram shows that peaks did move a bit relative to each other, as we can see by comparing the two sets of three circled peaks. In both cases, the upper chromatogram gives more even peak spacing between the peaks of interest. 

 Figure 1: Comparison of an "equivalent" gradient method at UHPLC and conventional LC pressures. Active ingredient (1) plus nine impurities (2)â(10). Adapted from reference 1. 

Back in 2005, when UHPLC was first becoming of commercial interest, we had a chance to examine one in our laboratory. In one of the tests we made, we ran a test mixture at several different pressures. These were run under what are considered equivalent gradient conditions. That is, the gradient volume, in terms of flow-rate times the gradient time, was constant, which should give equivalent separations. You can see that the four runs in Figure 2 all have the same gradient volume (0.4 mL/min X 15 min = 0.6 X 10 = 0.8 = 7.5 X 1.0 X 6 = 6 mL). With all other variables (mobile phase, temperature and so forth) held constant, we expected the pressure to change in proportion to the flow-rate, and this is what we saw. For example, at 1 mL/min, the pressure was 12 X 800 psi. At 0.8 mL/min, we would expect 0.8 X 12800 psi = 10240 psi, which is close to the observed 10400 psi. The same pattern was seen for the other flow-rates. Shorter retention times were expected with increased flow-rate and decreased gradient times, and this was observed, too. 

Figure 2: "Equivalent" gradient methods under conditions shown on chromatograms. 

What surprised me was that selectivity changed rather significantly. These separations were all done on the same column, with the same mobile phase, and under "equivalent gradient" conditions, so I expected to see the same separation in each case. But look closely, and you can see that this is not at all what happened. At 5400 psi, peaks 2 and 3 overlap completely. As pressure is increased, peak 2 moves forward, relative to peaks 1 and 3. I was excited about this and called a colleague, who calmly told me that pressure selectivity had been known since before I made my first injection. For example, in the 1960s Giddings

 pointed out that pressure changes could affect the molar volume of solutes and thus their interaction with the stationary phase. Realizing that I had not discovered something new, I filed the knowledge away in the gray matter and went about my business. Now and then I'd see an example, such as that in Figure 1, that I suspected was related to pressure selectivity. 

More recently, David McCalley's lab published two papers that dig deeper into the relationship of pressure and selectivity.

  They designed some simple experiments that examined pressure alone. This helps to unravel some of the ambiguity of the examples of Figures 1 and 2. In Figure 1, the column packing, flow-rate, gradient conditions and pressure were changed. In Figure 2, the flow-rate, gradient conditions and pressure were changed. In both of these cases, although "equivalent gradient" conditions can be argued, the interpretation of gradient data is a bit more difficult than with isocratic data. One other factor that may account for some of the changes observed in Figures 1 and 2 is an inadvertent change in column temperature when the flow-rate is changed. When an increase in flow-rate is made (all other conditions held constant), frictional heating of the column takes place, so the temperature of the column will increase. It is well known that temperature changes can affect selectivity, and this influence in addition to pressure can confuse interpretation of results, especially when other factors are changed simultaneously, such as for the results shown in Figures 1 and 2. 

Figure 3: Effect of pressure on selectivity. (1) thiourea; (2) propranolol; (3) diphenhydramine; (4) acetophenone; (5) protriptyline; (6), nitrobenzene. C18 column, acetonitrile/buffer (pH = 2.7). Adapted from reference 8.  

 In the two chromatograms of Figure 3, only the pressure is changed, so flow-rate and temperature variations are eliminated. This is done by placing a restriction capillary after the column, between the column and the detector. (The capillary has a negligible volume, and in back-up calculations, even this volume was taken into account.) Look at the dramatic difference between these two chromatograms — peak pairs (3–4) and peak pairs (5–6) both reverse when the pressure is increased from 43 to 739 bar (≈ 625 to ≈ 10700 psi). 

 looked at several different solute compounds, column stationary phases and mobile phases to try to sort out what is happening when pressure is changed. Here are some of the conclusions: 

1. In reversed-phase LC, retention increases for all molecules tested when pressure is increased. 

2. This effect of increased retention with increased pressure is true both for conventional silica-based columns as well as hybrid inorganic-organic based columns. The effect is more dramatic for C18 columns than for shorter-chain columns, but seems to be universal for reversed-phase. 

3. The water–organic ratio of the mobile phase affects the magnitude of the increase in retention with increased pressure. (This may confuse interpretation of gradient separations, such as those of Figures 1 and 2.) 

4. Retention increases with increased pressure were observed for all molecule types, but polar or ionized compounds were affected more strongly than non-polar ones of the same molecular weight, and larger molecules more than smaller ones of the same charge. In some cases, the retention changes were very large for a relatively small pressure increase. For example, prednisolone showed an increase in the retention factor, k, of ≈ 10%/100 bar (1450 psi) of pressure increase, although some other compounds changed by only 10% of this amount. 

5. For hydrophilic interaction chromatography (HILIC), the effects were the opposite of reversed-phase. That is, retention was reduced when pressure increased. 

Although the physical process of this phenomena is not completely understood, it is consistent with a change in the solute molecular volume — perhaps as the degree of hydration changes — when pressure is changed.  

From all of this I draw three important conclusions. Firstly, retention increases with increased pressure are universal in reversed-phase separations. Secondly, these changes can be of sufficient magnitude to be readily observed under pressure changes that may be encountered in the routine LC laboratory. Thirdly, different compounds and different states of ionization respond differently to the same change in pressure. 

How can we apply this information in a practical way to our day-to-day work? Most of us have never noticed changes in retention with pressure changes. One reason is that typical changes in pressure for a given LC method are relatively small. For example, as a column ages, the pressure may increase by 50 bar (725 psi) or so before we do something to mitigate the problem, such as replacing a frit or back-flushing the column. In this environment, small changes in selectivity might be just as well assigned to small changes in column chemistry as to pressure changes. When more dramatic changes in pressure are encountered, such as by a change in particle size (for example, Figure 1) or gradient (Figures 1 and 2), any small changes may be assigned to factors other than pressure. So even if pressure selectivity is present, we may not notice it or misinterpret the cause. 

Can we use pressure selectivity as a tool to control a separation, much as we discussed the use of solvent type, solvent strength, or column type in earlier articles? I think that this is a bit of a stretch at this point of our understanding of pressure selectivity. The relationships are not that well understood, the magnitude of retention changes can be small, and results may be unintentionally influenced by other factors, such as frictional heating of the mobile phase. As a result, I think it would be much better to invest our time in taking advantage of other selectivity variables instead.  

However, this is not to say that we can ignore pressure-retention effects. This is especially important when methods are used both in a UHPLC environment and a conventional-pressure LC environment. The separations of Figure 1 are a good example of this. In this case, UHPLC was used in the development laboratory because of its speed. But the routine laboratory lacked the instrumentation (and perhaps skills) to run this method in the UHPLC mode. For this reason, the method was scaled to conventional pressures (< 400 bar, 6000 psi) for routine use. This did not change the ability to apply the method for the intended application (nine impurities plus API), even though minor changes in selectivity were observed (circled peaks in Figure 1). What concerns me is the application to samples where all of the components are not known, such as a stability-indicating method with forcedegraded samples. For example, note the peaks in the top chromatogram of Figure 1 that I've marked with arrows. What has happened to these in the lower chromatogram? Did they drop below the detection limit, did they slide under neighbouring peaks because of a pressure-selectivity change, or...? Fortunately, in the present application it was not important, but it could be very important in other methods. 

I very much agree with one of McCalley's conclusions,

  "... an exact replication of the selectivity obtained when transferring methods for mixtures of different types of solute, developed with larger particle columns to small particle columns, should not necessarily be expected — even if the stationary phase particles differ only in their size." In other words, keep your eyes open for unexpected changes in peak spacing if you transfer methods from a high pressure system to a lower pressure one, or vice versa, if the change in pressure is more than ≈ 100 bar (≈ 1450 psi). Don't be caught by surprise! 

Strategies for Using Fast Liquid Chromatography in Method Development

, oral presentation at Pittsburgh Conference (2008). 

 is vice president of LC Resources, Walnut Creek, California. He is also a member of 

 editorial advisory board. Direct correspondence about this column should go to "LC Troubleshooting", 

, 4A Bridgegate Pavilion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or e-mail the editor, Alasdair Matheson, at 

Figure 1 was missing from the LC Troubleshooting article called "A Picture is Worth a Thousand Words" in the February issue. For an updated version of the article go to 

LC Troubleshooting

Selectivity in Reversed-Phase LC Separations (Part 4): Pressure Selectivity

The throughput of conventional separation systems is typically limited by the last eluting component of a mixture. In many applications most of the time is spent recording detector noise. A similar problem was encountered in the development of early continuous wave spectrometers, which was overcome by the introduction of transforms, such at the Fourier or Hadamard transforms. This article describes the use of information technology based on pseudo-random injection sequences derived from Hadamard matrices to perform rapid sample injections to increase the number of samples that can be analysed. This approach, called high-throughput multiplexing gas chromatography (htMPGC), offers a novel way to enhance the overall duty cycle, improve signal-to-noise ratio (

) and, ultimately, achieve extraordinarily high sample throughputs.

Screening of potential catalysts, drugs and lead structures or disease markers in medical diagnostics is of considerable scientific and economic interest.

  Parallelized high throughput assays are widely used to identify and quantify reaction products and determine conversions.

  Separation techniques play a dominant role in screening samples of high-throughput assays because highly complex compositions can be investigated with high precision and reproducibility. It is interesting to note that the bottle-neck in the screening of potential materials has shifted to the primary and — in particular — the secondary screening steps because sample preparation can be easily parallelized and automated to achieve high throughputs. Chromatographic methods are often time-consuming and limit the overall performance. Therefore great efforts have been made to improve the overall speed by optimizing continuous injections or using fast and parallelized separation techniques.

Another approach to enhance the analytical performance is the use of miniaturized separation devices, which minimize reagent consumption, reduce analysis time, provide good separation efficiency and detection sensitivity, and increase the overall sample throughput.

  However, the optimization of such systems can be time-consuming, especially for varying analyte concentrations. 

In the context of a secondary screening of potential catalysts, the feasibility of performing continuous real-time sampling in parallelized high-throughput assays is of great interest because detailed kinetic studies provide insights into activation and deactivation processes and long-term stabilities. Typically such investigations are performed in single-batch systems or samples from parallel reactors are sequentially analysed. This situation can be compared with continuous wave spectroscopy where the duty cycle is low and typically most of the acquisition time is spent recording detector noise.  

Multiplexing techniques — where multiple sources of information are combined and later mathematically deconvoluted — based on Fourier and Hadamard transformations,

  have completely changed this situation in spectroscopy

  to increase the duty cycle and improve the signal-to-noise ratio (

. This improvement is known as the Fellgett advantage.

  Previous attempts to apply multiplexing to separation techniques focused mainly on the 

The first report about the use of a transform technique in GC was published by Izawa et al. in 1967.

 This paper described a process gas chromatograph that continuously analysed injected gas samples (oxygen/nitrogen mixtures) using a pseudo-random binary signal generator. The output signal from the detector was gated by shifted input signals and filtered to continuously indicate the area of the corresponding chromatogram pattern of each component of interest. This experimental setup allowed simultaneous monitoring of several gas streams with only one separation column and because of the multiplexing advantage, the analysis could be performed more accurately and effectively. Obst

  developed a similar approach making use of the carrier gas pressure modulation according to a sinus function. Smit

  introduced pseudo-random binary sequences in trace analysis using GC and later also transferred this technique to LC.

 Here, sample injections were modulated with a 6-bit pseudo-random binary sequence (2

  – 1 = 63 elements) applying a time interval of 5 s between each element of the pseudo-random binary sequence. The gas chromatogram obtained was then crosscorrelated with the measured signal. 

In a series of publications Annino et al.

 explored multiplexing in GC to find optimum conditions for the effective use of correlation chromatography. An application of this technique is the continuous analysis of a process stream to measure small deviations. The pseudo-random binary modulation sequences between 10- and 11-bit (511 and 1023 elements, respectively) were generated by hard-wired correlators with a shift register and later by software, which gives a greater flexibility in the calculation. To minimize correlation noise caused by non-linearity of the distribution isotherms of an analyte between the mobile phase and the stationary phase, Phillips et al. developed a deconvolution algorithm based on the Fourier transform.

  Kaljurand and Küllik used pseudorandom injection pattern with a 6-bit sequence (63 elements) in GC to analyse polymers by pyrolysis in a continuous mode.

  Annino et al. and Küllik et al. reported in their publications that an optimal Fellgett advantage is achieved if the sample volume and concentration are low compared with conventional sample injection. Peak resolution typically was not changed by multiplexing. 

Early injection valves used for multiplexing in GC consisted of pneumatic valves to rapidly transfer the injection volume onto the separation column. Technical advances in this field make injections more precise and shorter injection times can be applied. Another type of injector for multiplexing GC was developed by Engelsma et al. based on a spark modulator.

  This modulator consists of a flow-through cell with two electrodes where a voltage of –5 to +5 kV is applied to generate a spark. The spark oxidizes the volatile organic compounds (VOCs) when air is used as carrier gas. Therefore the pseudo-random binary sequence controls the areas where the VOCs are erased and the oxidation products are not detected. Very recently, Lin et al. coupled Hadamard transform GC and Hadamard transform HPLC with MS.

  For the GC experiments, injections were controlled by an electromagnetic valve, while for the LC experiments an electronically controlled syringe pump in combination with a T-connector was used. Both of the injection devices permitted precise, successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After an inverse Hadamard transform of the encoded chromatogram, the 

of the signals were substantially improved compared with those expected from theoretical values. The 

was enhanced about 10-fold in Hadamard transform GC–MS and 6.8 in Hadamard transform LC–MS, using 10-bit and 11-bit modulation sequences, respectively. 

 Figure 1: (a) Conventional gas chromatogram of a gas mixture containing five components; (b) multiplexed gas chromatogram consisting of a convolution of the chromatogram depicted in (a). 

In information technology, efficient transmission of information from different sources is achieved by the mixing of signals and later this multiplexed information is deconvoluted to retrieve the individual information input. Based on this concept, we have recently developed high-throughput multiplexing chromatography to achieve very high sample throughputs (currently of up to 453 samples/h) by using structured modulation sequences which can also address single samples.

In contrast to a conventional separation [Figure 1(a)], where a sample is injected and the chromatogram is recorded until all analytes are eluted from the column, in multiplexing chromatography samples are continuously injected onto a column according to a pseudo-random sequence giving a convoluted chromatogram [Figure 1(b)]. 

Here, the experimental requirements, theory of operation and differences of MPGC and high-throughput multiplexing GC (htMPGC) are presented and discussed. 

All GC measurements were performed on a Focus GC (Thermo Fisher Scientific, San Jose, California, USA) equipped with a split injector and a flame ionization detector (FID) — both at 250 °C. The GC settings were controlled by the ChromCard software (Thermo Fisher Scientific). 

All separations were performed on a fused-silica column coated with GE-SE-30 (dimethylpolysiloxane) 25 m x 0.25 mm i.d., 0.5 µm film thickness (Macherey & Nagel, Düren, Germany) at 50 °C and 80 kPa head pressure. Helium was used as inert carrier gas and the split ratio was set to 1:200. Data was acquired at 10 Hz. 

The experimental setup consists of a laboratory-built onechannel or six-channel continuous-flow split/splitless injector with a heated sample port (250 °C), separately controlled by a fast 3/2 way solenoid valve (SMC corporation, Tokyo, Japan). The injector was manufactured from brass and is equipped with heating cartridges and a thermocouple to exactly control temperature. The sample ports are equipped with glass sleeves chemically deactivated with chlorotrimethylsilane. The multiplexing injectors used were directly mounted onto the split/splitless injector of the GC. 

Samples were continuously pumped through fused-silica capillaries (0.05 mm i.d.) into the sample port of the multiplexing injector and are continuously evaporated. The samples are repetitively injected into the split/splitless injector of the GC by short pressure pulses between 1 ms and 5 ms with He (400 kPa) according to the binary pseudo-random sequence. The multiplexing injector is purged through the injection needle by the split/splitless injector of the gas chromatograph. 

-bit binary pseudo-random sequences used throughout all experiments consist of 

 /2 elements '1', which are derived from an (

 + 1) Hadamard matrix. The modulation sequence was generated by a program written in Delphi (Embarcadero Technologies, South San Francisco, California, USA). Only for elements equal to 1 are sample injections performed. The modulation sequences were transmitted to a USB computer AD/DA converter interface. The signal of this is amplified by a transistor network and directly transmitted to the solenoid valves of the multiplexing injector. These signals are between 1–20 ms broad and open the solenoid valves for a pressure pulse to transfer the sample volume into the split/splitless injector of the Focus GC (Thermo Fisher Scientific). Data of the flame ionization detector (FID) was acquired at 10 Hz with the ChromCard software. The data acquisition was started by the software controlling the multiplexing injector. The data trace of the FID was converted into ASCII format for the data deconvolution. The Hadamard transformation and data deconvolution is performed by a program written in Delphi (Embarcadero Technologies). 

The experimental of multiplexing gas chromatography (MPGC) consists of a one-channel continuous-flow split/splitless injector, directly mounted onto the conventional split/splitless injector of a GC, with a heated sample port (250 °C), where small sample amounts are continuously evaporated and injected by short pressure pulses, which are controlled by a fast 3/2 way solenoid valve. To achieve a steady and stable sample flow, the sample is pumped by a syringe pump through a fused-silica capillary (50 µm, 2 m) into the sample port of the multiplexing injector. The samples are repetitively injected into the split/splitless injector of the GC by short pressure pulses of 2 ms with He (400 kPa) according to the binary pseudo-random sequence. The multiplexing injector is purged through the injection needle by the split/splitless injector of the gas chromatograph. 

Figure 2: Multiplexed gas chromatogram and Hadamard transformed gas chromatogram obtained by injection according to a 9-bit Hadamard sequence with a modulation time interval Ît = 1 s. 25 m GE-SE30 fs-capillary (i.d. 250 Âµm, film thickness 500 nm), isothermal separation conditions at 50 Â°C, 80 kPa inlet pressure, He as inert carrier gas.

-bit binary pseudo-random sequences consist of 

 + 1) Hadamard matrix. These sequences are constructed by the use of a virtual shift register applying logical operations. Sample injections are only performed for elements equal to 1. These signals are between 1–20 ms broad and open the solenoid valves for a pressure pulse to transfer the sample volume into the split/splitless injector of the GC. 

After the separation, the conventional chromatogram is obtained by Hadamard transformation of the 2

 /2 overlapping chromatograms according to the following equation:

The raw chromatogram is transformed into the circular representation which means that the time bins greater than the sequence length multiplied by the data acquisition rate are added to the beginning of the chromatogram. This chromatogram is then multiplied by the inverse simplex matrix obtained from the corresponding Hadamard matrix. 

In Figure 2 a multiplexed gas chromatogram obtained by injection according to a 9-bit Hadamard sequence with a modulation time interval 

This article describes a technique based on pseudo-random injection sequences to perform rapid sample injections.

LCGC Europe-04-01-2011

Is GMP Annex 11 Europe's Answer to 21 CFR 11?

On-line Whole Blood Analysis using Microextraction by Packed Syringe (MEPS) and LC–MS–MS

Selectivity in Reversed-Phase LC Separations (Part 4): Pressure Selectivity

High-throughput Multiplexing Gas Chromatography

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