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• From an analytical point of view, “quality” has always comprised two aspects. The qualitative side concerns establishing the identity of the material. The quantitative side is focused on measuring the amount of a given substance. Analytical chemistry is set up in a way that both qualitative and quantitative data can be provided for any known chemical substance.
  
• From the economic point of view, which is usually attached to synthetic products, quality is primarily associated with purity: the higher the better. Yet, depending on the manufacturing process or purpose of intended use, one might settle for a “lesser” quality and save money.
  
• Instances such as the thalidomide (Contergan) scandal and increasing numbers of lawsuits involving liability cases have driven the pharmaceutical industry into a situation where safety is the dominant issue. We are scared of the “unknown.” Quality=safety is now based on high purity and precisely measured dosage amount. Today, with any new drug comes a list of all possible impurities and a certificate that they are absent down to 0.05%. Chemists, pharmacists, lawyers, and regulators are in “total control” of the situation.

Talking about quality is now a much more diffuse issue. When we limit our view on plant-derived products and leave out mineral and animal-related products, quality begins with the right species. Next, a traceable connection must be established between the raw material, a defined manufacturing process, and the quality of the finished product. It must be determined what and how much of it becomes part of the product. This is what the FDA regulation concerning current good manufacturing practices (CGMP) for the botanical industry is trying to deal with.1

Unfortunately, this solves the problem of quality only in theory. These are some of the reasons for it:

• The framework of analytical approaches and legal consequences for quality control developed for (single) synthetic drugs cannot simply be applied to herbal drugs, which are complex mixtures of substances.
  
• The completely different philosophy behind the use and action of herbal drugs does not fit the mechanistic response model for isolated chemical substances. This makes it at least questionable as to why the same analytical tools (and approaches) should be applied.
  
• Unlike for synthetic drugs or isolated natural products, information about the composition of an herbal drug or preparation thereof is limited.
  
• There is no way to describe quantitatively the quality of a botanical drug unless the entire plant extract is regarded as the active component. Exceptions are a few cases where efficacy can be directly linked to a single or a combination of active components.

The higher the resolution and specificity of the chromatogram, the clearer small differences between samples will become. It is highly unlikely that two plants and consequently two finished products are exactly alike. Therefore, an analytical method must be chosen that allows identification with certainty but tolerates minor variations. Identification can be considered one of the dominating applications of HPTLC. One of the most appealing features of HPTLC fingerprints is the visual impression Fig 1–1. A broad spectrum of constituents can be detected and described without the need to know the chemical nature of each chromatogram zone.

Detection of Adulteration

One of the common problems in quality control of botanicals is the purposeful exchange or accidental mix-up of plant species. It affects quality in any case, but adulteration/falsification becomes critical if the wrong species is toxic. A popular case is Stephania, which is easily confused with various aristolochic acid—containing plant species such as Aristolochia fangji Fig 1–2. The requirements for a suitable method are specificity (being able to distinguish right from wrong species) and sufficient sensitivity for detecting small amounts of adulterant. HPTLC is well suited for these tasks, correlating good quality with the 4 absence of adulterants.

Figure 1–1 HPTLC fingerprint of various Ginkgo preparations.^* Mobile phase (MP): ethyl acetate, acetic acid, formic acid, water (100:11:11:26); derivatization (D): NP reagent, UV366 nm; tracks 1 and 2, ginkgo leaf; track 3, ginkgo leaf, freeze dried; tracks 4 to 7, ginkgo leaf extract, powder; track 8, rutin; tracks 9 and 10, ginkgo leaf extract, capsule; tracks 11 to 13, ginkgo leaf extract, tablet; track 14, ginkgo tincture. See Box 1–1 for discussion. *Note: Unless otherwise stated, all images in the book represent HPTLC plates Si 60 F254 (Merck, Darmstadt, Germany). All plates have been cropped below the application position and above the front. Only the tracks of interest are shown. For a list of abbreviations, see page 215.

Stability Tests

Stability tests on herbal drug preparations and finished products are a rather new application of HPTLC. During such tests, it has to be shown that a material does not change over the declared shelf life when stored properly. On the other hand, if any changes occur, they should be detectable. In this test, good quality is indicated by no change over time.

Figure 1–2 Detection of aristolochic acids as indication for adulteration. Mobile phase (MP), upper phase of toluene, ethyl acetate, water, formic acid (20:10:1:1); derivatization (D), tin (II) chloride reagent, UV 366 nm; 1 and 2, Aristolochia fangji, 1 and 10μ L; 3 and 4, aristolochic acids mix (Aldrich, St. Louis, MO), 10 and 50 ng; 5 and 6, Stephania tetrandra adulterated with 10% and 1% Aristolochia fangji, 10 μL; 7, pure Stephania tetrandra, 10μ L. See Box 1–2 for discussion.

The most commonly used measure of quality is related to the amount of certain marker compounds in a sample, even though “correct” quantities of the markers must not be confused with overall quality. Sufficient separation power of the chromatographic method to achieve baseline resolution for all components is usually desired. In many cases, quantitation is based on HPLC separation. It is often overlooked that with proper instrumentation and suitable methodology, quantitative determinations by HPTLC could give equal results at lower cost Fig 1–3.

In Chapter 4, we will discuss these applications in greater detail. Now we want to look at chromatography as a general method.

♦ What Is Chromatography?

One of the “scientific” definitions describes chromatography as “a separation technique based on the different affinities of the components of a mixture to two immiscible phases. One phase is stationary, the other mobile.”

Unfortunately, this definition doesn’t help much unless you really have a good background in physical chemistry, know about all sorts of equilibriums, and enjoy mathematical equations explaining why certain processes take place and others do not. If you want to brush up your knowledge in this regard, check out Refs. 2–4. Otherwise, let’s start with the model given in Fig 1–4.

A Simple Model of Chromatography

We want to separate a large group of people into subgroups of similar individuals and have invited volunteers to participate in an outdoor experiment. When looking closely at the participants, we notice that they seem to be very diverse and difficult to group as far as their interests are concerned. However, we can think about something basic that almost everybody would be attracted to: free food and drinks.

The rules of the experiment are easy:

1. Each participant sits in a boat anchored at the starting line across the river. At the signal, the clock is started, the anchors are lifted, and the boats are floating with the current toward the finish line. No sails and no paddles are allowed.
   
2. Vendors are providing free food and drinks on the banks of the river and have constructed docks for easy access to their facilities.
   
3. The participants can stop at the docks as often and as long as they desire. They are asked to get back into the boat and continue the journey when they have had enough.
   
4. At the finish line, the time and the number of arriving boats are recorded on a chart.

Figure 1–3 Quantitative determination of tetrandrine in Stephania tetrandra. (A) 3D plot of analog curves of samples (tracks 6 to 8) and standards (all other tracks). (B) Calibration function for tetrandrine. See Box 1–3 for discussion.

Here is what may happen: Most of the people will accept the invitation and have a good time eating. Some had enough for breakfast and don’t want to stop at all. They will arrive at the finish line shortly after the start of the game. Others may not be very hungry and soon realize that most vendors offer the same food. After a quick stop they continue, and it does not take very long for them to reach the goal. The hungry participants of course will feast, but finally most of them can’t eat any more, and so they arrive at the finish line. Of course, there are always a few people not paying attention, like the diver in the lower left corner of Fig 1–4.They just remain at the start line.

While everybody is having fun, the referee is creating a chart that looks like Fig 1–5.

Shortly after the start (t₀), a few boats will arrive. Their captains either forgot to stop or didn’t care. The time it took them (t_M) is dependent on the velocity of the river. The boats that arrive later will have stopped at least briefly, and the longer their captains ate and drank, the more time it took them (t_R1,t_R2, etc.) to finish. Certainly, the individual boats of each group are not coming in at exactly the same time. Just think about what happens if 50 hungry people enter the typical hamburger joint at one time; there will be some haggling. But also on the river there is some trouble: too many boats are trying to get going simultaneously. Some are pushed to the banks; others are getting caught at the docks without wanting to stop.

The whole set described in the previous section is called the chromatographic system. It could be for instance an HPLC column. The river is the mobile phase. It has a constant flow rate maintained by a pump. The mobile phase requires a certain time to move from one end of the system to the other. This is t_M, the mobile time. The clock starts when the sample is injected into the system. The docks can be viewed as the stationary phase to which the components of the sample have a certain affinity. There is also an affinity to the mobile phase. While in the mobile phase, the sample component is migrating. If a component interacts with the stationary phase, it is retained relative to those components, which are not interacting and arrive at t_M. The time differences are called net retention times (t_Rʹ). All molecules of a given sample component have the same affinity to the stationary phase and will be equally retained. With a suitable detector, the components arriving at the outlet of the column can be measured and recorded. The result is a chromatographic peak. Relative to the mobile time, each peak in the chromatogram can be assigned a capacity factor kʹ. This value is characteristic for a compound in a given chromatographic system and can be used for identification. More details on retention data are presented in Chapter 2. Unfortunately, such data are difficult to predict in general practice. That’s why qualitative chromatographic analysis is based on comparison to reference standards. If two compared samples are identical, they must have the same kʹ value, but the reverse relation is not true. That means even though two compounds are different, they may still have the same capacity factor. In this case, the chromatographic system is not selective. The trick is selecting an appropriate combination of stationary and mobile phase to achieve separation of a mixture.

To increase certainty that two substances are really identical, they are usually chromatographed in two or more chromatographic systems of different selectivity in which they must also have the same retention behavior. The other option is to subject the separated compound either online or off-line to other analytical methods such as ultraviolet (UV), infrared (IR), mass spectrometry (MS), or nuclear magnetic resonance (NMR) for so-called positive identification.

Figure 1–5 Retention in column chromatography (schematic): t₀, start of chromatogram (injection); t_M, mobile time (time for a nonretained molecule to pass the column); t_R, retention time (time it takes a retained molecule to pass the column); t_Rʹ, net retention time (retention time less mobile time) t_Rʹ = t_R–t_M; kʹ, capacity factor (a measure of retention independent of flow rate and column dimension) Kʹ=t_R–t_M/t_M.

Classification of Chromatography

The mobile phase of a chromatographic system can be a gas, a liquid, or a super-critical fluid. The stationary phase is either a solid or a liquid. These options allow convenient classification of the chromatographic process as listed in Table 1–1.

Some more specific details about the chromatographic principle will be given in Chapter 2. At the moment, the question about the “affinities” of sample components to the stationary phase and to the mobile phase shall be briefly addressed. The underlying interactions (separation principles) at the molecular level can be used to classify chromatography into several types (Table 1–2). The chromatographic system must be selected depending on the chemical nature of the components of a mixture.

So far, we have only discussed chromatography in a column, which is packed with small particles of the stationary phase. But there are other technical solutions, as seen in Table 1–3.

In planar chromatography, a somewhat different approach is taken. Coming back to our experiment on the river, we make a change. A certain time after the start of the event, the referee makes up his mind and calls everything off. Each participant has to remain at his or her current position, and a map is drawn to represent the actual situation. “Nonretained” participants will mark a “front” line, and others will be distributed at staggered positions along the way. Figure 1–6 illustrates the result schematically.

In planar chromatography, the stationary phase is a flat (thin) layer coated onto an inert support (i.e., glass, aluminum, or polyester). That’s where the term thin-layer chromatography comes from. There are of course many similarities between TLC and HPLC, because both are forms of LC. In general, the same stationary and mobile phases can be used with both techniques. The same kinds of samples are dealt with, and the performance is comparable. Today, both chromatographic methods offer mature high-performance systems at several levels of sophistication.

A difference between the methods is the way of expressing retention. In column chromatography, the components of a sample require individual times to pass the column. In other words, separation is based on variable retention times and fixed distance. In planar chromatography, the sample components are allowed to migrate for a certain time. This way, separation is based on variable migration distance and fixed time. Instead of the capacity factor kʹ of column chromatography, planar chromatography uses the R_F value to express relative retention.

Figure 1–6 Planar chromatogram (schematic): A, B, and C, various substances; MD, migration distance; zfʹ, distance of application position to solvent front; RF=MD/zfʹ

Another difference is associated with the mobile phase flow. In column chromatography, the mobile phase is maintained at a certain optimal velocity by means of gas pressure (GC) or pump action (HPLC). In TLC, capillary forces drive the mobile phase. In addition to stationary and mobile phases, TLC also takes advantage of a gas phase, which is usually present.

Column chromatography (we will consider only HPLC in the following) operates in online mode. Figure 1–7 gives a schematic overview including only the key elements. A pump continuously feeds the mobile phase from a reservoir through the column and the detector to waste. Capillary tubing makes all connections. With the help of an injector (sample loop), the sample is injected either manually or by an autosampler into the flow, reaches the column, and is separated. The individual components eluting from the column are measured in the detector and then put into waste. Other devices such as gradient mixers, column oven, and fraction collector (for preparative work) can also be connected online. For detection, UV, refractive index (RI), evaporative light scattering (ELSD), diode-array, fluorescence, electrochemical, or mass spectra detectors are available. The control of the entire system and handling of the data are typically computer based.

A key feature of the online principle is that multiple samples are analyzed in sequence. That means when the last component of an injection has left the chromatographic system, it must be reset before the next sample is injected. Operation can only be interrupted between samples, not during a run. While in the system, the sample is completely protected from environmental factors.

The sequence for a typical quantitative analysis consists of several calibration standards (if necessary in duplicate), a blank run, and any number of unknowns. If many samples are analyzed over a long period of time, control standards are included at certain intervals. As long as no changes are made to the system and the results for the control standard and the blank are within specification, the samples can be analyzed against the same calibration curve. Online operation is well suited for full automation.

Thin-layer chromatography uses the off-line principle (Fig 1–8). The process is separated into individual steps. First, the sample is applied onto the plate. The plate is then developed, if necessary derivatized, documented, and evaluated. The main difference to the online principle is that all steps are separated in time and location. Instruments (sample applicator, developing chamber, immersion device, documentation system, densitometer) are available for automation of individual steps. Even software controlling/managing the entire process can be employed. However, the plate as the central piece of TLC has to be moved manually through all steps. This may be seen as inferior to the fully automated HPLC setup, but as we will see, it adds tremendous flexibility to the TLC operation, which is unsurpassed by any other method.

TLC is the only chromatographic method offering the option of presenting the result as an image. A chromatogram can be seen as a sequence of dark, colored, or fluorescing zones on the plate, and it can be easily documented as a picture (Fig 1–9A).

An HPTLC chromatogram may lack the great sharpness of an HPLC separation, which is due to the higher efficiency of HPLC, but information can usually be communicated much better visually than in the form of verbal description or long peak tables. Just imagine how long it would take you to describe to someone else what you see in Fig 1–9B. How would you compare the eight samples on the plate in the form of analog curves or—to get the color information as well-in the form of 3D plots from a diode-array detector?

Figure 1–9 HPTLC fingerprints of feverfew and adulterants. Mobile phase (MP), cyclohexane, ethyl acetate (1:1). (A) UV 254 nm. (B) Derivatization (D), anisaldehyde reagent, white light; track 1, parthenolide; tracks 2 to 5, feverfew; track 6, Mexican feverfew; track 7, chamomile; track 8, Roman chamomile. See Box 1–4 for discussion.

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